Human TSLP DNA and polypeptides

ABSTRACT

The invention is directed to purified and isolated novel TSLP polypeptides, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and the uses of the above.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application hereby claims the benefit of United Statesprovisional application S. No. 60/108,452, filed Nov. 13, 1998, theentire disclosure of which is relied upon and incorporated by referenceherein.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention is directed to purified and isolated novel humanthymic stromal lymphopoietin (TSLP) pojypeptides and fragments thereof,the nucleic acids encoding such polypeptides, processes for productionof recombinant forms of such polypeptides, antibodies generated againstthese polypeptides, fragmented peptides derived from these polypeptides,and uses thereof

[0004] 2. Description of Related Art

[0005] Although B cell development has been extensively studied, therestill remain gaps in the pathway leading from hematopoeitic stem cellsto mature B cells. It is recognized that cytokines influence and play acritical role in B cell development and growth. Known cytokines thatinfluence B cell development include IL-2, IL-4, IL-5, IL-6, IL-7,IFN-gamma, and granulocyte-macrophage colony-stimulating factor(GM-CSF).

[0006] In recent years, a novel murine growth factor, designated thymicstromal lymphopoietin (TSLP), has been shown to play a role in B celldevelopment and maturation. The cytokine activity of murine TSLP is verysimilar to that of IL-7, which is required during proliferation andsurvival of pre-B cells (Janeway et al., Immuno Biology 2^(nd) Ed.(1996)). Both of these cytokines have been shown to sustain NAG8/7 cells(Friend et al., Exp. Hematol., 22:321-328 (1994)) and support Blymphopoiesis. In addition, mature B iymphocytes fail to develop in theabsence of either IL-7 or murine TSLP. Moreover, it has been shown thatmurine TSLP can replace IL-7 in sustaining B cell proliferativeresponses (Ray et al., Eur. J. Immunol., 26:10-16 (1996)). Thus, in themouse system, TSLP has a significant function in B cell development.

[0007] Like IL-7, murine TSLP can also costimulate thymocytes and matureT cells (Friend et al., Exp. Hematol., 22:321-328 (1994)). Studies withIL-7 receptor (IL-7R) knockout mice indicate that IL-7, TSLP, or bothplay a crucial role in controlling the rearrangement of the T cellreceptor-gamma (TCRy) locus, presumably by mediating accessibility ofthe TCRy genes to the VDJ recombinase (Candeias et al., ImmunologyLetters, 57:9-14 (1997)). Thus, murine TSLP also plays a significantrole in T-cell development.

[0008] Murine TSLP receptors and IL-7 receptors both use the IL-7Rα-chain as part of their signaling complexes (Levin et al., J. Immunol.,162:677-683 (1999)). Despite the common IL-7R a-chain, however, IL-7 andTSLP appear to mediate their lymphopoietic effects through distinctmechanisms. IL-7 induces activation of Stat5 and the Janus familykinases Jak1 and Jak3, whereas murine TSLP induces activation of Stat5,but not any of the known Janus family kinases (Levin et al., J.Immunol., 162:677-683 (1999)).

[0009] Given the important function of murine TSLP and the significanceof its role in B cell and T cell development and maturation in the mousesystem, there is a need in the art to identify and isolate human TSLPand to study its role in human B cell and T cell development andmaturation. In addition, in view of the continuing interest inlymphocyte development and the immune system, the discovery,identification, and roles of new proteins, such as human TSLP and itsreceptors, are at the forefront of modern molecular biology,biochemistry, and immunology. Despite the growing body of knowledge,there is still a need in the art for the identity and function ofproteins involved in cellular and immune responses.

[0010] In another aspect, the identification of the primary structure,or sequence, of an unknown protein is the culmination of an arduousprocess of experimentation. In order to identify an unknown protein, theinvestigator can rely upon a comparison of the unknown protein to knownpeptides using a variety of techniques known to those skilled in theart. For instance, proteins are routinely analyzed using techniques suchas electrophoresis, sedimentation, chromatography, sequencing and massspectrometry.

[0011] In particular, comparison of an unknown protein to polypeptidesof known molecular weight allows a determination of the apparentmolecular weight of the unknown protein (T. D. Brock and M. T. Madigan,Biology of Microorganisms, pp-76-77, Prentice Hall, 6d ed., (1991)).Protein molecular weight standards are commercially available to assistin the estimation of molecular weights of unknown protein (New EnglandBiolabs Inc. Catalog:130-131 (1995)); (J. L. Hartley, U.S. Pat. No.5,449,758). However, the molecular weight standards may not correspondclosely enough in size to the unknown protein to allow an accurateestimation of apparent molecular weight. The difficulty in estimation ofmolecular weight is compounded in the case of proteins that aresubjected to fragmentation by chemical or enzymatic means, modified bypost-translational modification or processing, and/or associated withother proteins in non-covalent complexes.

[0012] In addition, the unique nature of the composition of a proteinwith regard to its specific amino acid constituents results in uniquepositioning of cleavage sites within the protein. Specific fragmentationof a protein by chemical or enzymatic cleavage results in a unique“peptide fingerprint” (D. W. Cleveland et al., J. Biol. Chem.252:1102-1106 (1977); M. Brown et al., J. Gen. Virol. 50:309-316(1980)). Consequently, cleavage at specific sites results inreproducible fragmentation of a given protein into peptides of precisemolecular weights. Furthermore, these peptides possess unique chargecharacteristics that determine the isoelectric pH of the peptide. Theseunique characteristics can be exploited using a variety ofelectrophoretic and other techniques (T. D. Brock and M. T. Madigan,Biology of Microorganisms, pp. 76-77, Prentice Hall, 6d ed. (1991)).

[0013] Fragmentation of proteins is further employed for amino acidcomposition analysis and protein sequencing (P. Matsudiara, J. Biol.Chem., 262:10035-10038 (1987); C. Eckerskom et al., Electrophoresis,9:830-838 (1988)), particularly the production of fragments fromproteins with a “blocked” N-terminus. In addition, fragmented proteinscan be used for immunization, for affinity selection (R. A. Brown, U.S.Pat. No. 5,151,412), for determination of modification sites (e.g.phosphorylation), for generation of active biological compounds (T.D.Brock and M. T. Madigan, Biology of Microorganisms, 300-301 (PrenticeHall, 6d ed., (1991)), and for differentiation of homologous proteins(M. Brown et al., J. Gen. Virol., 50:309-316 (1980)).

[0014] In addition, when a peptide fingerprint of an unknown protein isobtained, it can be compared to a database of known proteins to assistin the identification of the unknown protein using mass spectrometry(W.J. Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015 (1993); D.Fenyo et al., Electrophoresis, 19:998-1005 (1998)). A variety ofcomputer software programs to facilitate these comparisons areaccessible via the Internet, such as Protein Prospector (Internet site:prospector.uscf edu), Multildent (Internet site:www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site:www.mann.embl-heiedelberg.de . . . deSearch/FR_PeptideSearch Form.html),and ProFound (Internet site:www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programsallow the user to specify the cleavage agent and the molecular weightsof the fragmented peptides within a designated tolerance. The programscompare these molecular weights to protein molecular weight informationstored in databases to assist in determining the identity of the unknownprotein. Accurate information concerning the number of fragmentedpeptides and the precise molecular weight of those peptides is requiredfor accurate identification. Therefore, increasing the accuracy indetermining of the number of fragmented peptides and the precisemolecular weight should result in enhanced likelihood of success in theidentification of unknown proteins.

[0015] In addition, peptide digests of unknown proteins can be sequencedusing tandem mass spectrometry (MS/MS) and the resulting sequencesearched against databases (J. K. Eng, et al., J. Am. Soc. Mass Spec.5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem., 66:4390-4399 (1994);J. A. Taylor and R. S. Johnson, Rapid Comm. Mass Spec., 11:1067-1075(1997)). Searching programs that can be used in this process exist onthe Internet, such as Lutefisk 97 (Internet site:www.lsbc.com:70/Lutefisk97.html), and the Protein Prospector, PeptideSearch and ProFound programs described above. Therefore, adding thesequence of a gene and its predicted protein sequence and peptidefragments to a sequence database can aid in the identification ofunknown proteins using tandem mass spectrometry.

[0016] Thus, there also exists a need in the art for polypeptidessuitable for use in peptide fragmentation studies, for use in molecularweight measurements, and for use in protein sequencing using tandem massspectrometry.

SUMMARY OF THE INVENTION

[0017] The invention aids in fulfilling these various needs in the artby providing isolated human TSLP nucleic acids and polypeptides encodedby these nucleic acids. Particular embodiments of the invention aredirected to an isolated TSLP nucleic acid molecule comprising the DNAsequence of SEQ ID NO:1 and an isolated TSLP nucleic acid moleculeencoding the amino acid sequence of SEQ ID NO:2, as well as nucleic acidmolecules complementary to these sequences. Both single-stranded anddouble-stranded RNA and DNA nucleic acid molecules are encompassed bythe invention, as well as nucleic acid molecules that hybridize to adenatured, double-stranded DNA comprising all or a portion of SEQ IDNO:1. Also encompassed are isolated nucleic acid molecules that arederived by in vitro mutagenesis of the nucleic acid molecule comprisingthe sequence of SEQ ID NO:1, that are degenerate from the nucleic acidmolecule comprising the sequence of SEQ ID NO:1, and that are allelicvariants of DNA of the invention. The invention also encompassesrecombinant vectors that direct the expression of these nucleic acidmolecules and host cells transformed or transfected with these vectors.

[0018] In addition, the invention encompasses methods of using thenucleic acid noted above to identify nucleic acids encoding proteinshaving the ability to induce B lineage or T lineage cell proliferation;to identify human chromosome number 5; to map genes on human chromosomenumber 5; to identify genes associated with certain diseases, syndromes,or other human conditions associated with human chromosome number 5; andto study cell signaling and the immune system.

[0019] The invention also encompasses the use of sense or antisenseoligonucleotides from the nucleic acid of SEQ ID NO:1 to inhibit theexpression of the polynucleotide encoded by the TSLP gene.

[0020] The invention also encompasses isolated polypeptides andfragments thereof encoded by these nucleic acid molecules includingsoluble polypeptide portions of SEQ ID NO:2. The invention furtherencompasses methods for the production of these polypeptides, includingculturing a host cell under conditions promoting expression andrecovering the polypeptide from the culture medium. Especially, theexpression of these polypeptides in bacteria, yeast, plant, insect, andanimal cells is encompassed by the invention.

[0021] In general, the polypeptides of the invention can be used tostudy cellular processes such as immune regulation, cell proliferation,cell differentiation, cell death, cell migration, cell-to-cellinteraction, and inflammatory responses. In addition, these polypeptidescan be used to identify proteins associated with TSLP ligands and TSLPreceptors.

[0022] In addition, the invention includes assays utilizing thesepolypeptides to screen for potential inhibitors of activity associatedwith polypeptide counter-structure molecules, and methods of using thesepolypeptides as therapeutic agents for the treatment of diseasesmediated by TSLP polypeptide counter-structure molecules. Further,methods of using these polypeptides in the design of inhibitors thereofare also an aspect of the invention.

[0023] The invention further includes a method for using thesepolypeptides as molecular weight markers that allow the estimation ofthe molecular weight of a protein or a fragmented protein, as well as amethod for the visualization of the molecular weight markers of theinvention thereof using electrophoresis. The invention furtherencompasses methods for using the polypeptides of the invention asmarkers for determining the isoelectric point of an unknown protein, aswell as controls for establishing the extent of fragmentation of aprotein.

[0024] Further encompassed by this invention are kits to aid in thesedeterminations.

[0025] Further encompassed by this invention is the use of the humanTSLP nucleic acid sequences, predicted amino acid sequences of thepolypeptide or fragments thereof, or a combination of the predictedamino acid sequences of the polypeptide and fragments thereof for use insearching an electronic database to aid in the identification of samplenucleic acids and/or proteins.

[0026] Isolated polyclonal or monoclonal antibodies that bind to thesepolypeptides are also encompassed by the invention, as well as the useof these antibodies to aid in purifying the TSLP polypeptide. Inaddition, the isolated antibodies can be used to establish anEnzyme-Linked Immunosorbent Assay (ELISA) to measure TSLP in samplessuch as serum.

BRIEF DESCRIPTION OF THE FIGURES

[0027]FIG. 1 presents the nucleotide sequence of human TSLP DNA (SEQ ID), and

[0028]FIG. 2 presents the amino acid sequence of human TSLP (SEQ IDNO:2).

DETAILED DESCRIPTION OF THE INVENTION

[0029] The nucleic acid molecules encompassed in the invention includethe wing nucleotide sequence:

[0030] Name: TSLP 1 GCAGCCAGAA AGCTCTGGAG CATCAGGGAG ACTCCAACTTAAGGCAACAG (SEQ ID NO:1) 51 CATGGGTGAA TAAGGGCTTC CTGTGGACTG GCAATGAGAGGCAAAACCTG 101 GTGCTTGAGC ACTGGCCCCT AAGGCAGGCC TTACAGATCT CTTACACTCG151 TGGTGGGAAG AGTTTAGTGT GAAACTGGGG TGGAATTGGG TGTCCACGTA 201TGTTCCCTTT TGCCTTACTA TATGTTCTCT CAGTTTCTTT CAGGAAAATC 251 TTCATCTTACAACTTGTAGG GCTGGTGTTA ACTTACGACT TCACTAACTG 301 TGACTTTGAG AAGATTAAAGCAGCCTATCT CAGTACTATT TCTAAAGACC 351 TGATTACATA TATGACTGGG ACCAAAAGTACCGAGTTCAA CAACACCGTC 401 TCTTGTAGCA ATCGGCCACA TTGCCTTACT GAAATCCAGAGCCTAACCTT 451 CAATCCCACC GCCCGCTGCG CGTCGCTCGC CAAAGAAATG TTCGCCATGA501 AAACTAAGGC TGCCTTAGCT ATCTGGTGCC CAGGCTATTC GGAAACTCAG 551ATAAATGCTA CTCAGGCAAT GAAGAAGAGG AGAAAAAGGA AAGTCACAAC 601 CAATAAATGTCTGGAACAAG TGTCACAATT ACAAGGATTG TGGCGTCGCT 651 TCAATCGACC TTTACTGAAACAACAGTAAA CCATCTTTAT TATGGTCATA 701 TTTCACAGCC CAAAATAAAT CATCTTTATTAAGTAAAAAA AAA

[0031] The amino acid sequence of the polypeptide encoded by thenucleotide sequence of the invention includes:

[0032] Name: TSLP (polypeptide) 1 MFPFALLYVL SVSFRKIFIL QLVGLVLTYDFTNCDFEKIK AAYLSTISKD (SEQ ID NO:2) 51 LITYMSGTKS TEFNNTVSCS NRPHCLTEIQSLTFNPTAGC ASLAKEMFAM 101 KTKAALAIWC PGYSETQINA TQAMKKRRKR KVTTNKCLEQVSQLQGLWRR 151 FNRPLLKQQ

[0033] The discovery of the nucleic acids of the invention enables theconstruction of expression vectors comprising nucleic acid sequencesencoding polypeptides; host cells transfected or transformed with theexpression vectors; isolated and purified biologically activepolypeptides and fragments thereof; the use of the nucleic acids oroligonucleotides thereof as probes to identify nucleic acid encodingproteins having TSLP-like activity (e.g., inducing B lineage or Tlineage cell proliferation), the use of the nucleic acids oroligonucleotides thereof to identify human chromosome number 5; the useof the nucleic acids or oligonucleotides thereof to map genes on humanchromosome number 5; the use of the nucleic acid or oligonucleotidesthereof to identify genes associated with certain diseases, syndromes orother human conditions associated with human chromosome number 5 and, inparticular, with the q21-q22 region of chromosome number 5, includingGardner syndrome, adenomatous polyposis coli, hereditary desmoiddisease, Turcot syndrome, and colorectal cancer, the use ofsingle-stranded sense or antisense oligonucleotides from the nucleicacids to inhibit expression of polynucleotides encoded by the TSLP gene;the use of such polypeptides and soluble fragments to induce B lineageor T lineage cell proliferation; the use of such polypeptides andfragmented peptides as molecular weight markers; the use of suchpolypeptides and fragmented peptides as controls for peptidefragmentation, and kits comprising these reagents; the use of suchpolypeptides and fragments thereof to generate antibodies; and the useof the antibodies to purify TSLP polypeptides.

[0034] Nucleic Acid Molecules

[0035] In a particular embodiment, the invention relates to certainisolated nucleotide sequences that are free from contaminatingendogenous material. A “nucleotide sequence” refers to a polynucleotidemolecule in the form of a separate fragment or as a component of alarger nucleic acid construct. The nucleic acid molecule has beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd sed., Cold SpringHarbor Laboratory, Cold Spring Hatbor, NY (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading firame,where the same do not interfere with manipulation or expression of thecoding region.

[0036] Nucleic acid molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, chemicallysynthesized DNA, DNA amplified by PCR, and combinations thereof. GenomicDNA may be isolated by conventional techniques, e.g., using the CDNA ofSEQ ID NO:1, or a suitable fragment thereof, as a probe.

[0037] The DNA molecules of the invention include fill length genes aswell as polynucleotides and fragments thereof. The full length gene mayalso include the N-terminal signal peptide. Other embodiments includeDNA encoding a soluble form, e.g., encoding the extracellular domain ofthe protein, either with or without the signal peptide.

[0038] The nucleic acids of the invention are preferentially derivedfrom human sources, but the invention includes those derived fromnon-human species, as well.

[0039] Preferred Sequences

[0040] The particularly preferred nucleotide sequence of the inventionis SEQ ID NO:1,as set forth above. A cDNA clone having the nucleotidesequence of SEQ ID NO:1 was isolated as described in Example 1. Thesequence of amino acids encoded by the DNA of SEQ ID NO:1 is shown inSEQ ID NO:2. This sequence identifies the TSLP polynucleotide as amember of a group of factors that influence the growth of B lineage andT lineage cells (Rayet al., Eur. J. Immunol, 26:10-16 (1996)); (Friendet al., Exp. Hematol., 22:321-328 (1994)).

[0041] Additional Sequences

[0042] Due to the known degeneracy of the genetic code, wherein morethan one codon can encode the same amino acid, a DNA sequence can varyfrom that shown in SEQ ID NO: 1, and still encode a polypeptide havingthe amino acid sequence of SEQ ID NO:2. Such variant DNA sequences canresult from silent mutations (e.g., occurring during PCR amplification),or can be the product of deliberate mutagenesis of a native sequence.

[0043] The invention thus provides isolated DNA sequences encodingpolypeptides of the invention, selected from: (a) DNA comprising thenucleotide sequence of SEQ ID NO:1; (b) DNA encoding the polypeptide ofSEQ ID NO:2; (c) DNA capable of hybridization to a DNA of (a) or (b)under conditions of moderate stringency and which encodes polypeptidesof the invention; (d) DNA capable of hybridization to a DNA of (a) or(b) under conditions of high stringency and which encodes polypeptidesof the invention, and (e) DNA which is degenerate as a result of thegenetic code to a DNA defined in (a), (b), (c), or (d) and which encodepolypeptides of the invention. Of course, polypeptides encoded by suchDNA sequences are encompassed by the invention.

[0044] As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth by(Sambrook et al. Molecular Cloning: A Laboratory Manual, 2ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989)), and includeuse of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5%SDS, 1.0 nM EDTA (pH 8.0), hybridization conditions of about 50%formamide, 6×SSC at about 42° C. (or other similar hybridizationsolution, such as Stark's solution, in about 50% formamide at about 42°C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS.Conditions of high stringency can also be readily determined by theskilled artisan based on, for example, the length of the DNA. Generally,such conditions are defined as hybridization conditions as above, andwith washing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilledartisan will recognize that the temperature and wash solution saltconcentration can be adjusted as necessary according to factors such asthe length of the probe.

[0045] Also included as an embodiment of the invention is DNA encodingpolypeptide fragments and polypeptides comprising inactivatedN-glycosylation site(s), inactivated protease processing site(s), orconservative amino acid substitution(s), as described below.

[0046] In another embodiment, the nucleic acid molecules of theinvention also comprise nucleotide sequences that are at least 80%identical to a native sequence. Also contemplated are embodiments inwhich a nucleic acid molecule comprises a sequence that is at least 90%identical, at least 95% identical, at least 98% identical, at least 99%identical, or at least 99.9% identical to a native sequence.

[0047] The percent identity may be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twonucleic acid sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described by(Devereux et al., Nucl. Acids Res., 12:387 (1984)) and available fromthe University of Wisconsin Genetics Computer Group (UWGCG). Thepreferred default parameters for the GAP program include: (1) a unarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix of(Gribskov and Burgess, Nucl. Acids Res., 14:6745 (1986)), as describedby (Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358 (1979)); (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps. Other programs usedby one skilled in the art of sequence comparison may also be used.

[0048] The invention also provides isolated nucleic acids useful in theproduction of polypeptides. Such polypeptides may be prepared by any ofa number of conventional techniques. A DNA sequence encoding a humanTSLP polypeptide, or desired fragment thereof may be subcloned into anexpression vector for production of the polypeptide or fragment. The DNAsequence advantageously is fused to a sequence encoding a suitableleader or signal peptide. Alternatively, the desired fragment may bechemically synthesized using known techniques. DNA fragments also may beproduced by restriction endonuclease digestion of a full length clonedDNA sequence, and isolated by electrophoresis on agarose gels. Ifnecessary, oligonucleotides that reconstruct the 5′ or 3′ terminus to adesired point may be ligated to a DNA fragment generated by restrictionenzyme digestion. Such oligonucleotides may additionally contain arestriction endonuclease cleavage site upstream of the desired codingsequence, and position an initiation codon (ATG) at the N-terminus ofthe coding sequence.

[0049] The well-known polymerase chain reaction (PCR) procedure also maybe employed to isolate and amplify a DNA sequence encoding a desiredprotein fragment. Oligonucleotides that define the desired termini ofthe DNA fragment are employed as 5′ and 3′ primers. The oligonucleotidesmay additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified DNA fragmentinto an expression vector. PCR techniques are described in (Saiki etal., Science, 239:487 (1988)); (Wu et al., Recombinant DNA Methodology,eds., Academic Press, Inc., San Diego, pp. 189-196 (1989)); and (Inniset al., PCR Protocols: A Guide to Methods and Applications, eds.,Academic Press, Inc. (1990)).

[0050] Polypeptides and Fragments Thereof

[0051] The invention encompasses polypeptides and fragments thereof invarious forms, including those that are naturally occurring or producedthrough various techniques such as procedures involving recombinant DNAtechnology. Such forms include, but are not limited to, derivatives,variants, and oligomers, as well as fusion proteins or fragmentsthereof.

[0052] Polypeptides and Fragments Thereof

[0053] The polypeptides of the invention include full length proteinsencoded by the nucleic acid sequences set forth above. Particularlypreferred polypeptides comprise the amino acid sequence of SEQ ID NO:2with particularly preferred fragments comprising amino acids 29 to 159(the mature polypeptide sequence) of SEQ ID NO:2.

[0054] The polypeptide of SEQ ID NO:2 includes an N-terminal hydrophobicregion that functions as a signal peptide. Computer analysis predictsthat the signal peptide corresponds to residues 1 to 28 of SEQ ID NO:2(although the next most likely computer-predicted signal peptidecleavage sites (in descending order) occur after amino acids 34 and 116of SEQ ID NO:2). Cleavage of the signal peptide thus would yield amature protein comprising amino acids 29 through 159 of SEQ ID NO:2.

[0055] The skilled artisan will recognize that the above-describedboundaries of such regions of the polypeptide are approximate. Toillustrate, the boundaries of the signal peptide (which may be predictedby using computer programs available for that purpose) may differ fromthose described above.

[0056] The polypeptides of the invention may be membrane bound or theymay be secreted and thus soluble. Soluble polypeptides are capable ofbeing secreted from the cells in which they are expressed. In general,soluble polypeptides may be identified (and distinguished fromnon-soluble membrane-bound counterparts) by separating intact cellswhich express the desired polypeptide from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired polypeptide. The presence of polypeptide in the mediumindicates that the polypeptide was secreted from the cells and thus is asoluble form of the protein.

[0057] In one embodiment, the soluble polypeptides and fragments thereofcomprise all or part of the extracellular domain, but lack thetransmembrane region that would cause retention of the polypeptide on acell membrane. A soluble polypeptide may include the cytoplasmic domain,or a portion thereof, as long as the polypeptide is secreted from thecell in which it is produced.

[0058] Other embodiments include soluble fragments having an N-terminusat amino acids 29 or 35 and a C-terminus at amino acid 159.

[0059] In general, the use of soluble forms is advantageous for certainapplications. Purification of the polypeptides from recombinant hostcells is facilitated, since the soluble polypeptides are secreted fromthe cells. Further, soluble polypeptides are generally more suitable forintravenous administration.

[0060] The invention also provides polypeptides and fragments of theextracellular domain that retain a desired biological activity.Particular embodiments are directed to polypeptide fragments that retainthe ability to bind TSLP receptors. Such a fragment may be a solublepolypeptide, as described above. In another embodiment, the polypeptidesand fragments advantageously include regions that are conserved amongthe family of proteins that influence the growth of B lineage or Tlineage cells described above.

[0061] Also provided herein are polypeptide fragments comprising atleast 20, or at least 30, contiguous amino acids of the sequence of SEQID NO:2. Fragments derived from the cytoplasmic domain find use instudies of signal transduction and in regulating cellular processesassociated with transduction of biological signals. Polypeptidefragments also may be employed as immunogens, in generating antibodies.

[0062] Variants

[0063] Naturally occurring variants as well as derived variants of thepolypeptides and fragments are provided herein.

[0064] Variants may exhibit amino acid sequences that are at least 80%identical. Also contemplated are embodiments in which a polypeptide orfragment comprises an amino acid sequence that is at least 90%identical, at least 95% identical, at least 98% identical, at least 99%identical, or at least 99.9% identical to the preferred polypeptide orfragment thereof. Percent identity may be determined by visualinspection and mathematical calculation. Alternatively, the percentidentity of two protein sequences can be determined by comparingsequence information using the GAP computer program, based on thealgorithm of (Needleman and Wunsch, J. Mol. Bio., 48:443 (1970)) andavailable from the University of Wisconsin Genetics Computer Group(UVGCG). The preferred default parameters for the GAP program include:(1) a scoring matrix, blosum62, as described by (Henikoff and HenikoffProc. Natl. Acad. Sci. USA, 89:10915 (1992)); (2) a gap weight of 12;(3) a gap length weight of 4; and (4) no penalty for end gaps. Otherprograms used by one skilled in the art of sequence comparison may alsobe used.

[0065] The variants of the invention include, for example, those thatresult from alternate mRNA splicing events or from proteolytic cleavage.Alternate splicing of mRNA may, for example, yield a truncated butbiologically active protein, such as a naturally occurring soluble formof the protein. Variations attributable to proteolysis include, forexample, differences in the N- or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the protein (generally from 1-5 terminal amino acids).Proteins in which differences in amino acid sequence are attributable togenetic polymorphism (allelic variation among individuals producing theprotein) are also contemplated herein.

[0066] Additional variants within the scope of the invention includepolypeptides that may be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives may be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein, as discussed in more detail below.

[0067] Other derivatives include covalent or aggregative conjugates ofthe polypeptides with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.Examples of fusion proteins are discussed below in connection witholigomers. Further, fusion proteins can comprise peptides added tofacilitate purification and identification. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in (Hopp et al., Bio/Technology, 6:1204(1988)). One such peptide is the FLAG® peptide,Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, (SEQ ID NO:3) which is highly antigenicand provides an epitope reversibly bound by a specific monoclonalantibody, enabling rapid assay and facile purification of expressedrecombinant protein. A murine hybridoma designated 4E11 produces amonoclonal antibody that binds the FLAG® peptide in the presence ofcertain divalent metal cations, as described in U.S. Pat. No. 5,011,912,hereby incorporated by reference. The 4E11 hybridoma cell line has beendeposited with the American Type Culture Collection under accession no.HB 9259. Monoclonal antibodies that bind the FLAG® peptide are availablefrom Eastman Kodak Co., Scientific Imaging Systems Division, New Haven,Conn.

[0068] Among the variant polypeptides provided herein are variants ofnative polypeptides that retain the native biological activity or thesubstantial equivalent thereof. One example is a variant that binds withessentially the same binding affinity as does the native form. Bindingaffinity can be measured by conventional procedures, e.g., as describedin U.S. Pat. No. 5,512,457 and as set forth below.

[0069] Variants include polypeptides that are substantially homologousto the native form, but which have an amino acid sequence different fromthat of the native form because of one or more deletions, insertions orsubstitutions. Particular embodiments include, but are not limited to,polypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequence.

[0070] A given amino acid may be replaced, for example, by a residuehaving similar physiochemical characteristics. Examples of suchconservative substitutions include substitution of one aliphatic residuefor another, such as lie, Val, Leu, or Ala for one another;substitutions of one polar residue for another, such as between Lys andArg, Glu and Asp, or Gin and Asn; or substitutions of one aromaticresidue for another, such as Phe, Trp, or Tyr for one another. Otherconservative substitutions, e.g., involving substitutions of entireregions having similar hydrophobicity characteristics, are well known.

[0071] Similarly, the DNAs of the invention include variants that differfrom a native DNA sequence because of one or more deletions, insertionsor substitutions, but that encode a biologically active polypeptide.

[0072] The invention further includes polypeptides of the invention withor without associated native-pattern glycosylation. Polypeptidesexpressed in yeast or mammalian expression systems (e.g., COS-1 or COS-7cells) can be similar to or significantly different from a nativepolypeptide in molecular weight and glycosylation pattern, dependingupon the choice of expression system. Expression of polypeptides of theinvention in bacterial expression systems, such as E. coli, providesnon-glycosylated molecules. Further, a given preparation may includemultiple differentially glycosylated species of the protein. Glycosylgroups can be removed through conventional methods, in particular thoseutilizing glycopeptidase. In general, glycosylated polypeptides of theinvention can be incubated with a molar excess of glycopeptidase(Boehringer Mannheim).

[0073] Correspondingly, similar DNA constructs that encode variousadditions or substitutions of amino acid residues or sequences, ordeletions of terminal or internal residues or sequences are encompassedby the invention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid and Y is Ser or Thr. Appropriate substitutions, additions, ordeletions to the nucleotide sequence encoding these triplets will resultin prevention of attachment of carbohydrate residues at the Asn sidechain. Alteration of a single nucleotide, chosen so that Asn is replacedby a different amino acid, for example, is sufficient to inactivate anN-glycosylation site. Alternatively, the Ser or Thr can by replaced withanother amino acid, such as Ala. Known procedures for inactivatingN-glycosylation sites in proteins include those described in U.S. Pat.No. 5,071,972 and EP 276,846, hereby incorporated by reference.

[0074] In another example of variants, sequences encoding Cys residuesthat are not essential for biological activity can be altered to causethe Cys residues to be deleted or replaced with other amino acids,preventing formation of incorrect intramolecular disulfide bridges uponfolding or renaturation.

[0075] Other variants are prepared by modification of adjacent dibasicamino acid residues, to enhance expression in yeast systems in whichKEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

[0076] Oligomers

[0077] Encompassed by the invention are oligomers or fusion proteinsthat contain human TSLP polypeptides. Such oligomers may be in the formof covalently-linked or non-covalently-linked multimers, includingdimers, trimers, or higher oligomers. As noted above, preferredpolypeptides are soluble and thus these oligomers may comprise solublepolypeptides. In one aspect of the invention, the oligomers maintain thebinding ability of the polypeptide components and provide therefor,bivalent, trivalent, etc., binding sites.

[0078] One embodiment of the invention is directed to oligomerscomprising multiple polypeptides joined via covalent or non-covalentinteractions between peptide moieties fused to the polypeptides. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of the polypeptides attached thereto, asdescribed in more detail below.

[0079] Immunoglobulin-Based Oligomers

[0080] As one alternative, an oligomer is prepared using polypeptidesderived from immunoglobulins. Preparation of fusion proteins comprisingcertain heterologous polypeptides fused to various portions ofantibody-derived polypeptides (including the Fe domain) has beendescribed, e.g., by (Ashkenazi et al., PNAS USA, 88:10535 (1991)); (Bymet al., Nature, 344:677 (1990)); and (Hollenbaugh and Aruffo“Construction of Immunoglobulin Fusion Proteins”, in Current Protocolsin Immunology, Suppl. 4, pp. 10.19.1-10.19.11(1992)).

[0081] One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing a polypeptide of theinvention to an Fe polypeptide derived from an antibody. A gene fusionencoding the polypeptide/Fc fusion protein is inserted into anappropriate expression vector. Polypeptide/Fc fusion proteins areexpressed in host cells transformed with the recombinant expressionvector, and allowed to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fe moieties to yielddivalent molecules.

[0082] The term “Fc polypeptide” as used herein includes native andmutein forms of polypeptides made up of the Fe region of an antibodycomprising all of the CH domains of the Fc region. Truncated forms ofsuch polypeptides containing the hinge region that promotes dimerizationare also included. Preferred polypeptides comprise an Fc polypeptidederived from a human IgG1 antibody.

[0083] One suitable Fe polypeptide, described in PCT application WO93/10151 (hereby incorporated by reference), is a single chainpolypeptide extending from the N-terminal hinge region to the nativeC-terminus of the Fe region of a human IgG1 antibody. Another useful Fepolypeptide is the Fe mutein described in U.S. Pat. No. 5,457,035 and in(Baum et al., EMBO J, 13:39924001 (1994)) incorporated herein byreference. The amino acid sequence of this mutein is identical to thatof the native Fe sequence presented in WO 93/10151, except that aminoacid 19 has been changed from Leu to Ala, amino acid 20 has been changedfrom Leu to Glu, and amino acid 22 has been changed from Gly to Ala. Themutein exhibits reduced affinity for Fe receptors.

[0084] The above-described fusion proteins comprising Fe moieties (andoligomers formed therefrom) offer the advantage of facile purificationby affinity chromatography over Protein A or Protein G columns.

[0085] In other embodiments, the polypeptides of the invention may besubstituted for the variable portion of an antibody heavy or lightchain. If fusion proteins are made with both heavy and light chains ofan antibody, it is possible to form an oligomer with as many as fourTSLP extracellular regions.

Peptide-Linker Based Oligomers

[0086] Alternatively, the oligomer is a fusion protein comprisingmultiple polypeptides, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233, which are hereby incorporated byreference. A DNA sequence encoding a desired peptide linker may beinserted between, and in the same reading frame as, the DNA sequences ofthe invention, using any suitable conventional technique. For example, achemically synthesized oligonucleotide encoding the linker may beligated between the sequences. In particular embodiments, a fusionprotein comprises from two to four soluble TSLP polypeptides, separatedby peptide linkers.

[0087] Leucine-Zippers

[0088] Another method for preparing the oligomers of the inventioninvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., Science 240:1759 (1988)), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize.

[0089] The zipper domain (also referred to herein as an oligomerizing,or oligomer-forming, domain) comprises a repetitive heptad repeat, oftenwith four or five leucine residues interspersed with other amino acids.Examples of zipper domains are those found in the yeast transcriptionfactor GCN4 and a heat-stable DNA-binding protein found in rat liver(C/EBP; Landschulz et al., Science, 243:1681 (1989)). Two nucleartransforming proteins, fos and jun, also exhibit zipper domains, as doesthe gene product of the murine proto-oncogene, c-myc (Landschulz et al.,Science, 240:1759 (1988)). The products of the nuclear oncogenes fos andjun comprise zipper domains that preferentially form heterodimer (O'Sheaet al., Science, 245:646 (1989)), (Turner and Tjian, Science, 243:1689(1989)). The zipper domain is necessary for biological activity (DNAbinding) in these proteins.

[0090] The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess zipper domains (Buckland and Wild, Nature, 338:547 (1989);(Britton, Nature, 353:394 (1991)); (Delwart and Mosialos, AIDS Researchand Human Retroviruses; 6:703 (1990)). The zipper domains in thesefusogenic viral proteins are near the transmembrane region of theproteins; it has been suggested that the zipper domains could contributeto the oligomeric structure of the fusogenic proteins. Oligomerizationof fusogenic viral proteins is involved in fusion pore formation (Spruceet al, Proc. Natl. Acad. Sci. U.S. 88:3523 (1991)). Zipper domains havealso been recently reported to play a role in oligomerization ofheat-shock transcription factors (Rabindran et al., Science 259:230(1993)).

[0091] Zipper domains fold as short, parallel coiled coils. (O'Shea etal., Science 254:539 (1991)). The general architecture of the parallelcoiled coil has been well characterized, with a “knobs-into-holes”packing as proposed by (Crick, Acta Crystallogr., 6:689)). The dimerformed by a zipper domain is stabilized by the heptad repeat, designated(abcdefg)_(n) according to the notation of (McLachlan and Stewart, J.Mol. Biol., 98:293 (1975)), in which residues a and d are generallyhydrophobic residues, with d being a leucine, which line up on the sameface of a helix. Oppositely-charged residues commonly occur at positionsg and e. Thus, in a parallel coiled coil formed from two helical zipperdomains, the “knobs” formed by the hydrophobic side chains of the firsthelix are packed into the “holes” formed between the side chains of thesecond helix.

[0092] The residues at position d (often leucine) contribute largehydrophobic stabilization energies, and are important for oligomerformation (Krystek et al., Int. J. Peptide Res., 38:229 (1991)).(Lovejoy et al., Science 259:1288 (1993)) recently reported thesynthesis of a triple-stranded a-helical bundle in which the helices runup-up-down. Their studies confirmed that hydrophobic stabilizationenergy provides the main driving force for the formation of coiled coilsfrom helical monomers. These studies also indicate that electrostaticinteractions contribute to the stoichiometry and geometry of coiledcoils. Further discussion of the stricture of leucine zippers is foundin (Harbury et al., Science, 262:1401 (Nov. 26, 1993)).

[0093] Examples of leucine zipper domains suitable for producing solubleoligomeric proteins are described in PCT application WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in (Hoppe et al., FEBS Letters, 344:191 (1994)), herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in (Fanslow et al., Semin. Immunol., 6:267-278 (1994)).Recombinant fusion proteins comprising a soluble polypeptide fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomer that forms is recovered from the culture supernatant.

[0094] Certain leucine zipper moieties preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in (Hoppe et al., FEBS Letters, 344:191 (1994)) andin U.S. Pat. No. 5,716,805, hereby incorporated by reference in theirentirety. This lung SPD-derived leucine zipper peptide comprises theamino acid sequence Pro Asp Val Ala Ser Leu Arg Gin Gin Val GIu Ala LeuGln Gly Gin Val Gin His Leu Gin Ala Ala Phe Ser GIn Tyr (SEQ ID NO: 4).

[0095] Another example of a leucine zipper that promotes trimerizationis a peptide comprising the amino acid sequence Arg Met Lys Gin lie GluAsp Lys Ile Gin Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu lie AlaArg lie Lys Lys Leu lie Gly Glu Arg, (SEQ ID NO: 5), as described inU.S. Pat. No. 5,716,805. In one alternative embodiment, an N-terminalAsp residue is added; in another, the peptide lacks the N-terminal Argresidue.

[0096] Fragments of the foregoing zipper peptides that retain theproperty of promoting oligomerization may be employed as well. Examplesof such fragments include, but are not limited to, peptides lacking oneor two of the N-terminal or C-terminal residues presented in theforegoing amino acid sequences. Leucine zippers may be derived fromnaturally occurring leucine zipper peptides, e.g., via conservativesubstitution(s) in the native amino acid sequence, wherein the peptide'sability to promote oligomerization is retained.

[0097] Other peptides derived from naturally occurring trimeric proteinsmay be employed in preparing trimeric oligomers. Alternatively,synthetic peptides that promote oligomerization may be employed. Inparticular embodiments, leucine residues in a leucine zipper moiety arereplaced by isoleucine residues. Such peptides comprising isoleucine maybe referred to as isoleucine zippers, but are encompassed by the term“leucine zippers” as employed herein.

[0098] Production of Polypeptides and Fragments Thereof

[0099] Expression, isolation and purification of the polypeptides andfragments of the invention may be accomplished by any suitabletechnique, including but not limited to the following:

[0100] Expression Systems

[0101] The present invention also provides recombinant cloning andexpression vectors containing DNA, as well as host cell containing therecombinant vectors. Expression vectors comprising DNA may be used toprepare the polypeptides or fragments of the invention encoded by theDNA. A method for producing polypeptides comprises culturing host cellstransformed with a recombinant expression vector encoding thepolypeptide, under conditions that promote expression of thepolypeptide, then recovering the expressed polypeptides from theculture. The skilled artisan will recognize that the procedure forpurifying the expressed polypeptides will vary according to such factorsas the type of host cells employed, and whether the polypeptide ismembrane-bound or a soluble form that is secreted from the host cell.

[0102] Any suitable expression system may be employed. The vectorsinclude a DNA encoding a polypeptide or fragment of the invention,operably linked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, microbial,viral, or insect gene. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, an MRNA ribosomalbinding site, and appropriate sequences which control transcription andtranslation initiation and termination. Nucleotide sequences areoperably linked when the regulatory sequence functionally relates to theDNA sequence. Thus, a promoter nucleotide sequence is operably linked toa DNA sequence if the promoter nucleotide sequence controls thetranscription of the DNA sequence. An origin of replication that confersthe ability to replicate in the desired host cells, and a selection geneby which transformants are identified, are generally incorporated intothe expression vector.

[0103] In addition, a sequence encoding an appropriate signal peptide(native or heterologous) can be incorporated into expression vectors. ADNA sequence for a signal peptide (secretory leader) may be fused inframe to the nucleic acid sequence of the invention so that the DNA isinitially transcribed, and the MRNA translated, into a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells promotes extracellular secretion of thepolypeptide. The signal peptide is cleaved from the polypeptide uponsecretion of polypeptide from the cell.

[0104] The skilled artisan will also recognize that the position(s) atwhich the signal peptide is cleaved may differ from that predicted bycomputer program, and may vary according to such factors as the type ofhost cells employed in expressing a recombinant polypeptide. A proteinpreparation may include a mixture of protein molecules having differentN-terminal amino acids, resulting from cleavage of the signal peptide atmore than one site. Particular embodiments of mature proteins providedherein include, but are not limited to, proteins having the residue atposition 16, 29, 35, 95, or 117 of SEQ ID NO:2 as the N-terminal aminoacid.

[0105] Suitable host cells for expression of polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cellsare generally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in (Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985)). Cell-freetranslation systems could also be employed to produce polypeptides usingRNAs derived from DNA constructs disclosed herein.

[0106] Prokaryotic Systems

[0107] Prokaryotes include gram-negative or gram-positive organisms.Suitable prokaryotic host cells for transformation include, for example,E. coli, Bacillus subtilis, Salmonella typhimurium, and various otherspecies within the genera Pseudonionas, Streptomyces, andStaphylococcus. In a prokaryotic host cell, such as E. coli, apolypeptide may include an N-terminal methionine residue to facilitateexpression of the recombinant polypeptide in the prokaryotic host cell.The N-terminal Met may be cleaved from the expressed recombinantpolypeptidc.

[0108] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and a DNAsequence are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA).

[0109] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615 (1978); and (Goeddel etal., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddelet al., Nucl. Acids Res. 8:4057 (1980); and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412 (1982)). A particularly useful prokaryotic host cellexpression system employs a phage λP_(L)promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of theλP_(L) promoter include plasmid pHUB2 (resident in E. coli strain JMB9,ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

[0110] Yeast Systems

[0111] Alternatively, the polypeptides may be expressed in yeast hostcells, preferably from the Saccharomyces genus (e.g., S. cerevisiae).Other genera of yeast, such as Pichia or Kluyveronzyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg. 7:149 (1968)); and (Holland et al., Biochem. 17:4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in (Hitzeman, EPA-73,657). Anotheralternative is the glucose-repressible ADH2 promoter described by(Russell et al., J. Biol. Chem. 258:2674 (1982)) and (Beier et al.,Nature 300:724 (1982)). Shuttle vectors replicable in both yeast and E.coli may be constructed by inserting DNA sequences from pBR322 forselection and replication in E. coli (Ampr gene and origin ofreplication) into the above described yeast vectors.

[0112] The yeast α-factor leader sequence may be employed to directsecretion of the polypeptide. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., (Kujan et al., Cell 30:933 (1982)) and (Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330 (1984)). Other leader sequences suitablefor facilitating secretion of recombinant polypeptides from yeast hostsare known to those of skill in the art. A leader sequence may bemodified near its 3′ end to contain one or more restriction sites. Thiswill facilitate fusion of the leader sequence to the structural gene.

[0113] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by (Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929 (1978)). The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 mg/ml adenine and 20 mg/ml uracil.

[0114] Yeast host cells transformed by vectors containing an ADH2promoter sequence may be grown for inducing expression in a “rich”medium. An example of a rich medium is one consisting of 1% yeastextract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenineand 80 mg/ml uracil. Derepression of the ADH2 promoter occurs whenglucose is exhausted from the medium.

[0115] Mammalian or Insect Systems

[0116] Mammalian or insect host cell culture systems also may beemployed to express recombinant polypeptides. Bacculovirus systems forproduction of heterologous proteins in insect cells are reviewed by(Luckow and Summers, Bio/Technology, 6:47 (1988)). Established celllines of mammalian origin also may be employed. Examples of suitablemammalian host cell lines include the COS-7 line of monkey kidney cells(ATCC CRL 1651) (Gluzman et al., Cell 23:175 (1981)), L cells, C127cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLacells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell linederived from the African green monkey kidney cell line CV1 (ATCC CCL 70)as described by (McMahan et al., EMBO J., 10: 2821 (1991)).

[0117] Established methods for introducing DNA into mammalian cells havebeen described (Kaufman, R. J., Large Scale Mammalian Cell Culture, pp.15-69 (1990)). Additional protocols using commercially availablereagents, such as Lipofectamine lipid reagent (Gibco/BRL) orLipofectamine-Plus lipid reagent, can be used to transfect cells(Felgner et al., Proc. Natl. Aad. Sci. USA 84:7413-7417 (1987)). Inaddition, electroporation can be used to transfect mammalian cells usingconventional procedures, such as those in (Sambrook et al., MolecularCloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring HarborLaboratory Press (1989)). Selection of stable transformants can beperformed using methods known in the art, such as, for example,resistance to cytotoxic drugs. (Kaufman et al., Meth. in Enzymology185:487-511 (1990)), describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B 11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980)). Aplasmid expressing the DHFR CDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

[0118] Transcriptional and translational control sequences for mammalianhost cell expression vectors can be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113 (1978)); (Kaufman, Meth. inEnzymology (1990)). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

[0119] Additional control sequences shown to improve expression ofheterologous genes from mammalian expression vectors include suchelements as the expression augmenting sequence element (EASE) derivedfrom CHO cells (Morris et al., Animal Cell Technology, pp. 529-534 andPCT Application WO 97/25420 (1997)) and the tripartite leader (TPL) andVA gene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem.257:13475-13491 (1982)). The internal ribosome entry site (IRES)sequences of viral origin allows dicistronic mRNAs to be translatedefficiently (Oh and Samow, Current Opinion in Genetics and Development3:295-300 (1993)); (Ramesh et al., Nucleic Acids Research 24:2697-2700(1996)). Expression of a heterologous cDNA as part of a dicistronic mRNAfollowed by the gene for a selectable marker (e.g. DHFR) has been shownto improve transfectability of the host and expression of theheterologous cDNA (Kaufman, Meth. in Enzymology (1990)) Exemplaryexpression vectors that employ dicistronic mRNAs are pTR-DC/GFPdescribed by (Mosser et al., Biotechniques 22:150-161 (1997)), and p2A5Idescribed by (Morris et al., Animal Cell Technology, pp. 529-534(1997)).

[0120] A useful high expression vector, pCAVNOT, has been described by(Mosley et al., Cell 59:335-348 (1989)). Other expression vectors foruse in mammalian host cells can be constructed as disclosed by (Okayamaand Berg, Mol. Cell. Biol. 3:280 (1983)). A useful system for stablehigh level expression of mammalian cDNAs in C127 murine mammaryepithelial cells can be constructed substantially as described by(Cosman et al., Mol. Immunol. 23:935 (1986)). A useful high expressionvector, PMLSV N1/N4, described by (Cosman et al., Nature 312:768(1984)), has been deposited as ATCC 39890. Additional useful mammalianexpression vectors are described in EP-A-0367566, and in WO 91/18982,incorporated by reference herein. In yet another alternative, thevectors can be derived from retroviruses.

[0121] Another useful expression vector, pFLAG, can be used. FLAG®technology is centered on the fusion of a low molecular weight (1 kD),hydrophilic, FLAG® marker peptide to the N-terminus of a recombinantprotein expressed by pFLAG® expression vectors. pDC311 is anotherspecialized vector used for expressing proteins in CHO cells. pDC311 ischaracterized by a bicistronic sequence containing the gene of interestand a dihydrofolate reductase (DHFR) gene with an internal ribosomebinding site for DHFR translation, an expression augmenting sequenceelement (EASE), the human CMV promoter, a tripartite leader sequence,and a polyadenylation site.

[0122] Regarding signal peptides that may be employed, the native signalpeptide may be replaced by a heterologous signal peptide or leadersequence, if desired. The choice of signal peptide or leader may dependon factors such as the type of host cells in which the recombinantpolypeptide is to be produced. To illustrate, examples of heterologoussignal peptides that are functional in mammalian host cells include thesignal sequence for interleukin-7 (IL-7) described in U.S. Pat. No.4,965,195; the signal sequence for interleukin-2 receptor described in(Cosman et al., Nature 312:768 (1984)); the interleukin-4 receptorsignal peptide described in EP 367,566; the type I interleukin-1receptor signal peptide described in U.S. Pat. No. 4,968,607; and thetype II interleukin-1 receptor signal peptide described in EP 460,846.

[0123] Purification

[0124] The invention also includes methods of isolating and purifyingthe polypeptides and fragments thereof.

[0125] Isolation and Purification

[0126] The “isolated” polypeptides or fragments thereof encompassed bythis invention are polypeptides or fragments that are not in anenvironment identical to an environment in which it or they can be foundin nature. The “purified” polypeptides or fragments thereof encompassedby this invention are essentially free of association with otherproteins or polypeptides, for example, as a purification product ofrecombinant expression systems such as those described above or as apurified product from a non-recombinant source such as naturallyoccurring cells and/or tissues.

[0127] In one preferred embodiment, the purification of recombinantpolypeptides or fragments can be accomplished using fusions ofpolypeptides or fragments of the invention to another polypeptide to aidin the purification of polypeptides or fragments of the invention. Suchfusion partners can include the poly-His or other antigenicidentification peptides described above as well as the Fe moietiesdescribed previously.

[0128] With respect to any type of host cell, as is known to the skilledartisan, procedures for purifying a recombinant polypeptide or fragmentwill vary according to such factors as the type of host cells employedand whether or not the recombinant polypeptide or fragment is secretedinto the culture medium.

[0129] In general, the recombinant polypeptide or fragment can beisolated from the host cells if not secreted, or from the medium orsupernatant if soluble and secreted, followed by one or moreconcentration, salting-out, ion exchange, hydrophobic interaction,affinity purification or size exclusion chromatography steps. As tospecific ways to accomplish these steps, the culture medium first can beconcentrated using, a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. In addition, achromatofocusing step can be employed. Alternatively, a hydrophobicinteraction chromatography step can be employed. Suitable matrices canbe phenyl or octyl moieties bound to resins. In addition, affinitychromatography with a matrix which selectively binds the recombinantprotein can be employed. Examples of such resins employed are lectincolumns, dye columns, and metal-chelating columns. Finally, one or morereversed-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, (e.g., silica gel or polymer resinhaving pendant methyl, octyl, octyldecyl or other aliphatic groups) canbe employed to further purify the polypeptides. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide an isolated and purified recombinantprotein.

[0130] It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the invention.

[0131] In this aspect of the invention, polypeptide-binding proteins,such as the anti-polypeptide antibodies of the invention or otherproteins that may interact with the polypeptide of the invention, can bebound to a solid phase support such as a column chromatography matrix ora similar substrate suitable for identifying, separating, or purifyingcells that express polypeptides of the invention on their surface.Adherence of polypeptide-binding proteins of the invention to a solidphase contacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

[0132] Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first can be incubatedwith a biotinylated polypeptide-binding protein of the invention.Incubation periods are typically at least one hour in duration to ensuresufficient binding to polypeptides of the invention. The resultingmixture then is passed through a column packed with avidin-coated beads,whereby the high affinity of biotin for avidin provides the binding ofthe polypeptide-binding cells to the beads. Use of avidin-coated beadsis known in the art. See (Berenson, et al. J. Cell. Biochem., 10D:239(1986)). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

[0133] The desired degree of purity depends on the intended use of theprotein. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no protein bands correspondingto other proteins are detectable upon analysis by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). It will be recognized by one skilled in thepertinent field that multiple bands corresponding to the polypeptide maybe visualized by SDS-PAGE, due to differential glycosylation,differential post-translational processing, and the like. Mostpreferably, the polypeptide of the invention is purified to substantialhomogeneity, as indicated by a single protein band upon analysis bySDS-PAGE. The protein band may be visualized by silver staining,Coomassie blue staining, or (if the protein is radiolabeled) byautoradiography.

[0134] Assays

[0135] The purified polypeptides of the invention (including proteins,polypeptides, fragments, variants, oligomers, and other forms) may betested for the ability to bind TSLP receptors in any suitable assay,such as a conventional binding assay. To illustrate, the polypeptide maybe labeled with a detectable reagent (e.g., a radionuclide, chromophore,enzyme that catalyzes a colorimetric or fluorometric reaction, and thelike). The labeled polypeptide is contacted with cells expressing TSLPreceptors. The cells then are washed to remove unbound labeledpolypeptide, and the presence of cell-bound label is determined by asuitable technique, chosen according to the nature of the label.

[0136] One example of a binding assay procedure is as follows. Arecombinant expression vector containing TSLP cDNA is constructed bymethods known in the art. The mouse TSLP receptor comprises anN-terminal extracellular domain, a transmembrane region, and aC-terminal cytoplasmic domain. CV1-EBNA-1 cells in 10 cm² dishes aretransfected with the recombinant expression vector. CV-1/EBNA-1 cells(ATCC CRL 10478) constitutively express EBV nuclear antigen-i drivenfrom the CMV immediate-early enhancer/promoter. CV1-EBNA-1 was derivedfrom the African Green Monkey kidney cell line CV-1 (ATCC CCL 70), asdescribed by (McMahan et al., EMBO J. 10:2821 (1991)).

[0137] The transfected cells are cultured for 24 hours, and the cells ineach dish then are split into a 24-well plate. After culturing anadditional 48 hours, the transfected cells (about 4×10⁴ cells/well) arewashed with BM-NFDM, which is binding medium (RPMI 1640 containing 25mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) towhich 50 mg/ml nonfat dry milk has been added. The cells then areincubated for 1 hour at 37° C. with various concentrations of, forexample, a soluble polypeptide/Fc fusion protein made as set forthabove. Cells then are washed and incubated with a constant saturatingconcentration of a ¹²⁵I-mouse anti-human IgG in binding medium, withgentle agitation for 1 hour at 37° C. After extensive washing, cells arereleased via trypsinization.

[0138] The mouse anti-human IgG employed above is directed against theFc region of human IgG and can be obtained from Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa. The antibody is radioiodinated usingthe standard chloramine-T method. The antibody will bind to the Fcportion of any polypeptide/Fc protein that has bound to the cells. Inall assays, non-specific binding of ¹²⁵I-antibody is assayed in theabsence of the Fc fusion protein/Fc, as well as in the presence of theFc fusion protein and a 200-fold molar excess of unlabeled mouseanti-human IgG antibody.

[0139] Cell-bound ¹²⁵I-antibody is quantified on a Packard Autogammacounter. Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51:660(1949)) are generated on RS/1 (BBN Software, Boston, Mass.) run on aMicrovax computer.

[0140] Another type of suitable binding assay is a competitive bindingassay. To illustrate, biological activity of a variant may be determinedby assaying for the variant's ability to compete with the native proteinfor binding to TSLP receptors.

[0141] Competitive binding assays can be performed by conventionalmethodology. Reagents that may be employed in competitive binding assaysinclude radiolabeled TSLP and intact cells expressing TSLP receptors(endogenous or recombinant) on the cell surface. For example, aradiolabeled soluble TSLP fragment can be used to compete with a solubleTSLP variant for binding to cell surface TSLP receptors. Instead ofintact cells, one could substitute a soluble TSLP receptor/Fc fusionprotein bound to a solid phase through the interaction of Protein A orProtein G (on the solid phase) with the Fc moiety. Chromatographycolumns that contain Protein A and Protein G include those availablefrom Pharmacia Biotech, Inc., Piscataway, N.J.

[0142] Another type of competitive binding assay utilizes radiolabeledsoluble TSLP receptor, such as a soluble TSLP receptor/Fc fusionprotein, and intact cells expressing endogenous or recombinant TSLPreceptor. The radiolabeled TSLP receptor can be used to compete with themembrane bound TSLP receptor for soluble TSLP. Qualitative results canbe obtained by competitive autoradiographic plate binding assays, whileScatchard plots (Scatchard, Ann. N.Y. Acad. Sci. 51:660 (1949)) may beutilized to generate quantitative results.

[0143] Use of Human TSLP Nucleic Acid or Oligonucleotides

[0144] In addition to being used to express polypeptides as describedabove, the nucleic acids of the invention, including DNA, RNA, mRNA andoligonucleotides thereof can be used:

[0145] as probes to identify nucleic acid encoding proteins having theability to induce B lineage or T lineage cell proliferation;

[0146] to identify human chromosome number 5;

[0147] to map genes on human chromosome number 5;

[0148] to identify genes associated with certain diseases, syndromes, orother conditions associated with human chromosome number 5;

[0149] as single-stranded sense or antisense oligonucleotides, toinhibit expression of polypeptide encoded by the TSLP gene;

[0150] to help detect defective genes in an individual; and

[0151] for gene therapy.

[0152] Probes

[0153] Among the uses of nucleic acids of the invention is the use offragments as probes or primers. Such fragments generally comprise atleast about 17 contiguous nucleotides of a DNA sequence. In otherembodiments, a DNA fragment comprises at least 30, or at least 60,contiguous nucleotides of a DNA sequence.

[0154] Because homologs of SEQ ID NO: 1, from other mammalian species,are contemplated herein, probes based on the human DNA sequence of SEQID NO:1 may be used to screen cDNA libraries derived from othermammalian species, using conventional cross-species hybridizationtechniques.

[0155] Using knowledge of the genetic code in combination with the aminoacid sequences set forth above, sets of degenerate oligonucleotides canbe prepared. Such oligonucleotides are useful as primers, e.g., inpolymerase chain reactions (PCR), whereby DNA fragments are isolated andamplified.

[0156] Chromosome Mapping

[0157] All or a portion of the nucleic acids of SEQ ID NO:1, includingoligonucleotides, can be used by those skilled in the art usingwell-known techniques to identify the human chromosome 5, and thespecific locus thereof, that may contain the DNA of other TSLP familymembers. Useful techniques include, but are not limited to, using thesequence or portions, including oligonucleotides, as a probe in variouswell-known techniques such as radiation hybrid mapping (highresolution), in situ hybridization to chromosome spreads (moderateresolution), and Southern blot hybridization to hybrid cell linescontaining individual human chromosomes (low resolution).

[0158] For example, chromosomes can be mapped by using PCR and radiationhybridization PCR is performed using the Whitehead Institute/MIT Centerfor Genome Research Genebridge4 panel of 93 radiation hybrids(http://www-genome.wi.mit.edu/ftp/distribution/human-STS-releases/july97/rhmap/genebridge4.html).Primers are used which lie within a putative exon, across an intron, oracross an intron-exon fragment of the gene of interest and which amplifya product from human genomic DNA, but do not amplify, for example,control hamster genomic DNA. The results of the PCRs are converted intoa data vector that is submitted to the Whitehead/MIT Radiation Mappingsite on the internet (http://www-seq.wi.mit.edu). The data is scored andthe chromosomal assignment and placement relative to known Sequence TagSite (STS) markers on the radiation hybrid map is provided. Thefollowing web site provides additional information about radiationhybrid mapping:(http://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/07-97.1NTRO.html).

[0159] Identifying Associated Diseases

[0160] As set forth below, SEQ ID NO:1 has been mapped to the q21-q22region of chromosome 5 by syntenic analysis of the murine gene. Thus,the nucleic acid of SEQ ID NO:1 or a fragment thereof can be used by oneskilled in the art using well-known techniques to analyze abnormalitiesassociated with human chromosome number 5 and, in particular, with theq21-q22 region of chromosome number 5, including Gardner syndrome,adenomatous polyposis coli, hereditary desmoid disease, Turcot syndrome,and colorectal cancer. This enables one to distinguish conditions inwhich this marker is rearranged or deleted. In addition, nucleotides ofSEQ ID NO:1 or a fragment thereof can be used as a positional marker tomap other genes of unknown location.

[0161] The DNA may be used in developing treatments for any disordermediated (directly or indirectly) by defective or insufficient amountsof the genes corresponding to the nucleic acids of the invention.Disclosure herein of native nucleotide sequences permits the detectionof defective genes, and the replacement thereof with normal genes.Defective genes may be detected in in vitro diagnostic assays, and bycomparison of a native nucleotide sequence disclosed herein with that ofa gene derived from a person suspected of harboring a defect in thisgene.

[0162] Sense-Antisense

[0163] Other useful fragments of the nucleic acids include antisense orsense oligonucleotides comprising a single-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target mRNA (sense)or DNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of DNA (SEQ IDNO: 1). Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to about 30 nucleotides. Theability to derive an antisense or a sense oligonucleotide, based upon aCDNA sequence encoding a given protein is described in, for example,(Stein and Cohen, Cancer Res. 48:2659 (1988)) and (van der Krol et al.,BioTechniques 6:958 (1988)).

[0164] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that block orinhibit protein expression by one of several means, including enhanceddegradation of the mRNA by RNAseH, inhibition of splicing, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression ofproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO91/06629) and whereinsuch sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

[0165] Other examples of sense or antisense oligonucleotides includethose oligonucleotides which are covalently linked to organic moieties,such as those described in WO 90/10448, and other moieties thatincreases affinity of the oligonucleotide for a target nucleic acidsequence, such as poly-(L-lysine). Further still, intercalating agents,such as ellipticine, and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

[0166] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, lipofection, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus.

[0167] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0168] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0169] Use of Human TSLP Polypeptides and Fragmented Polypeptides

[0170] Uses include, but are not limited to, the following:

[0171] Purifying proteins and measuring activity thereof

[0172] Delivery Agents

[0173] Therapeutic and Research Reagents

[0174] Molecular weight and Isoelectric focusing markers

[0175] Controls for peptide fragmentation

[0176] Identification of unknown proteins

[0177] Preparation of Antibodies

[0178] Purification Reagents

[0179] The polypeptide of the invention finds use as a proteinpurification reagent. For example, the polypeptides may be used topurify TSLP binding partners, such as human TSLP receptors. Inparticular embodiments, a polypeptide (in any form described herein thatis capable of binding TSLP receptors) is attached to a solid support byconventional procedures. As one example, affinity chromatography columnscontaining functional groups that will react with functional groups onamino acid side chains of proteins are available (Pharmacia Biotech,Inc., Piscataway, N.J.). In an alternative, a TSLP polypeptide/Fcprotein (as discussed above) is attached to Protein A- or ProteinG-containing chromatography columns through interaction with the Fcmoiety.

[0180] The polypeptide also finds use in purifying or identifying cellsthat express TSLP receptors on the cell surface. Polypeptides are boundto a solid phase such as a column chromatography matrix or a similarsuitable substrate. For example, magnetic microspheres can be coatedwith the polypeptides and held in an incubation vessel through amagnetic field. Suspensions of cell mixtures containing TSLP receptorexpressing cells are contacted with the solid phase having thepolypeptides thereon. Cells expressing TSLP receptor on the cell surfacebind to the fixed polypeptides, and unbound cells then are washed away.

[0181] Alternatively, the polypeptides can be conjugated to a detectablemoiety, then incubated with cells to be tested for TSLP receptorexpression. After incubation, unbound labeled matter is removed and thepresence or absence of the detectable moiety on the cells is determined.

[0182] In a further alternative, mixtures of cells suspected ofcontaining TSLP receptors are incubated with biotinylated polypeptides.Incubation periods are typically at least one hour in duration to ensuresufficient binding. The resulting mixture then is passed through acolumn packed with avidin-coated beads, whereby the high affinity ofbiotin for avidin provides binding of the desired cells to the beads.Procedures for using avidin-coated beads are known (see Berenson, et al.J. Cell. Biochem., 10D:239 (1986)). Washing to remove unbound material,and the release of the bound cells, are performed using conventionalmethods.

[0183] Measuring Activity

[0184] Polypeptides also find use in measuring the biological activityof TSLP receptors in terms of their binding affinity. The polypeptidesthus may be employed by those conducting “quality assurance” studies,e.g., to monitor shelf life and stability of protein under differentconditions. For example, the polypeptides may be employed in a bindingaffinity study to measure the biological activity of a TSLP receptorthat has been stored at different temperatures, or produced in differentcell types. The proteins also may be used to determine whetherbiological activity is retained after modification of a TSLP receptor(e.g., chemical modification, truncation, mutation, etc.). The bindingaffinity of the modified TSLP receptor is compared to that of anunmodified TSLP receptor to detect any adverse impact of themodifications on biological activity of TSLP receptors. The biologicalactivity of a TSLP receptor thus can be ascertained before it is used ina research study, for example.

[0185] Delivery Agents

[0186] The polypeptides also find use as carriers for delivering agentsattached thereto to cells bearing TSLP receptors. Cells expressing TSLPreceptors include those identified in thymus, spleen, kidney, and bonemarrow. The polypeptides thus can be used to deliver diagnostic ortherapeutic agents to such cells (or to other cell types found toexpress TSLP receptors on the cell surface) in in vitro or in vivoprocedures.

[0187] Detectable (diagnostic) and therapeutic agents that may beattached to a polypeptide include, but are not limited to, toxins, othercytotoxic agents, drugs, radionuclides, chromophores, enzymes thatcatalyze a colorinmetric or fluorometric reaction, and the like, withthe particular agent being chosen according to the intended application.Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonasaeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins suchas trichothecenes, and derivatives and fragments (e.g., single chains)thereof. Radionuclides suitable for diagnostic use include, but are notlimited to, ¹²³I, ¹³¹I, ^(99m)Tc, ¹¹¹In, and ⁷⁶Br. Examples ofradionuclides suitable for therapeutic use are ¹³¹I, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re,¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu.

[0188] Such agents may be attached to the polypeptide by any ableconventional procedure. The polypeptide comprises functional groups onamino acid side chains that can be reacted with functional groups on adesired agent to form covalent bonds, for example. Alternatively, theprotein or agent may be derivatized to generate or attach a desiredreactive functional group. The derivatization may involve attachment ofone of the bifunctional coupling reagents available for attachingvarious molecules to proteins (Pierce Chemical Company, Rockford, Ill.).A number of techniques for radiolabeling proteins are known.Radionuclide metals may be attached to polypeptides by using a suitablebifunctional chelating agent, for example.

[0189] Conjugates comprising polypeptides and a suitable diagnostic ortherapeutic agent (preferably covalently linked) are thus prepared. Theconjugates are administered or otherwise employed in an amountappropriate for the particular application.

[0190] Therapeutic Agents

[0191] Polypeptides of the invention may be used in developingtreatments for any disorder mediated (directly or indirectly) bydefective, or insufficient amounts of the polypeptides. Thesepolypeptides may be administered to a mammal afflicted with such adisorder.

[0192] The polypeptides may also be employed in inhibiting thebiological activity of TSLP receptors in in vitro or in vivo procedures.For example, a purified or modified polypeptide or a fragment thereof(e.g., modified TSLP polypeptides that bind the receptor but lack theability to induce signaling) may be used to inhibit binding ofendogenous TSLP to cell surface receptors. Biological effects thatresult from the binding of endogenous TSLP to receptors thus areinhibited.

[0193] In addition, TSLP receptor polypeptides may be administered to amammal to treat a TSLP receptor-mediated disorder. Such TSLPreceptor-mediated disorders include conditions caused (directly orindirectly) or exacerbated by TSLP receptors.

[0194] Compositions of the present invention may contain a polypeptidein any form described herein, such as native proteins, variants,derivatives, oligomers, and biologically active fragments. In particularembodiments, the composition comprises a soluble TSLP polypeptide or anoligomer comprising soluble TSLP polypeptides.

[0195] Compositions comprising an effective amount of a polypeptide ofthe present invention, in combination with other components such as aphysiologically acceptable diluent, carrier, or excipient, are providedherein. The polypeptides can be formulated according to known methodsused to prepare pharmaceutically useful compositions. They can becombined in admixture, either as the sole active material or with otherknown active materials suitable for a given indication, withpharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate,and phosphate buffered solutions), preservatives (e.g., thimerosal,benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable formulations for pharmaceutical compositions includethose described in (Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Company, Easton, Pa. (1980)).

[0196] In addition, such compositions can be complexed with polyethyleneglycol (PEG), metal ions, or incorporated into polymeric compounds suchas polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., orincorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance, and are thuschosen according to the intended application.

[0197] The compositions of the invention can be administered in anysuitable manner, e.g., topically, parenterally, or by inhalation. Theterm “parenteral” includes injection, e.g., by subcutaneous,intravenous, or intramuscular routes, also including localizedadministration, e.g., at a site of disease or injury. Sustained releasefrom implants is also contemplated. One skilled in the pertinent artwill recognize that suitable dosages will vary, depending upon suchfactors as the nature of the disorder to be treated, the patient's bodyweight, age, and general condition, and the route of administration.Preliminary doses can be determined according to animal tests, and thescaling of dosages for human administration is performed according toart-accepted practices.

[0198] Compositions comprising nucleic acids in physiologicallyacceptable formulations are also contemplated. DNA may be formulated forinjection, for example.

[0199] Research Agents

[0200] Another use of the polypeptide of the present invention is as aresearch tool for studying the biological effects that result frominhibiting TSLP/FSLP receptor interactions on different cell types.Polypeptides also may be employed in in vitro assays for detecting TSLPor TSLP receptors or the interactions thereof.

[0201] Another embodiment of the invention relates to uses of human TSLPto study B cell or T cell signal transduction. Human TSLP and othercytokines play a central role in B cell and T cell development andimmune responses, including transducing cellular signals, stimulatingcells to secrete cytokines, and inducing B cell and T cellproliferation. As such, alterations in the expression and/or activationof TSLP can have profound effects on a plethora of cellular processes,including, but not limited to, activation or inhibition of cell specificresponses and proliferation. Expression of cloned TSLP or ofcatalytically inactive mutants of TSLP has been used to identify therole a particular protein plays in mediating specific signaling events.

[0202] Cellular signaling often involves a molecular activation cascade,during which a receptor propagates a ligand-receptor mediated signal byspecifically activating intracellular kinases which phosphorylate targetsubstrates. These substrates can themselves be kinases which becomeactivated following phosphorylation. Alternatively, they can be adaptormolecules that facilitate down stream signaling through protein-proteininteraction following phosphorylation. Regardless of the nature of thesubstrate molecule(s), expressed catalytically active versions of theTSLP ligand receptors can be used to identify what substrate(s) wererecognized and activated by the TSLP ligand receptor(s). As such, thesenovel TSLP receptors can be used as reagents to identify novel moleculesinvolved in signal transduction pathways.

[0203] In addition, TSLP can be used by one skilled in the art usingwell-known techniques to stimulate B lineage or T lineage cellproliferation (Ray et al., Eur. J. Immunology 26, 10-16 (1996)) and(Namikawa et al., Blood 87:1881-1890 (1996)), to expression clone thehuman TSLP receptor (Sims et al., Science 241:585-589 (1988)), to clonea related protein (Kozlosky et. al., Cytokine 9:540-549 (1997)) and(Lyman et al., Blood 10:2795-2801 (1994)), and to ex vivo expand cells(Piacibello et al., Blood 89:2644-2653 (1997)).

[0204] Uses Thereof

[0205] Thus, the present invention encompasses methods of stimulating B-and T-lymphocyte proliferation, where the method comprises incubatinglymphocytes with human TSLP. In a further embodiment, the methodcomprises incubating lymphocytes with human TSLP and at least one othercytokine in vivo or in vitro. Preferably, the cytokine, is selected fromthe group of IL-7, Steel Factor, Stem Cell Factor, Mast Cell GrowthFactor or flt3-Ligand. More preferably the cytokine is IL-7.

[0206] The present invention also encompasses methods of stimulatinglymphocyte development or lymphopoiesis, where the method comprisesincubating progenitor cells, such as bone marrow-derived mononuclearcells, with human TSLP in vivo or in vitro. In a further embodiment, themethod comprises incubating lymphocytes with human TSLP and at least oneother cytokine. Preferably, the cytokine is selected from the group ofIL-7, Steel Factor, Stem Cell Factor, Mast Cell Growth Factor orflt3-Ligand. More preferably the cytokine is IL-7.

[0207] Molecular Weight and Isoelectric Point Markers

[0208] The polypeptides of the present invention can be subjected tofragmentation into smaller peptides by chemical and enzymatic means, andthe peptide fragments so produced can be used in the analysis of otherproteins or polypeptides. For example, such peptide fragments can beused as peptide molecular weight markers, peptide isoelectric pointmarkers, or in the analysis of the degree of peptide fragmentation.Thus, the invention also includes these polypeptides and peptidefragments, as well as kits to aid in the determination of the apparentmolecular weight and isoelectric point of an unknown protein and kits toassess the degree of fragmentation of an unknown protein.

[0209] Although all methods of fragmentation are encompassed by theinvention, chemical fragmentation is a preferred embodiment, andincludes the use of cyanogen bromide to cleave under neutral or acidicconditions such that specific cleavage occurs at methionine residues (E.Gross, Methods in Enz. 11:238-255 (1967)). This can further includeadditional steps, such as a carboxymethylation step to convert cysteineresidues to an unreactive species.

[0210] Enzymatic fragmentation is another preferred embodiment, andincludes the use of a protease such as Asparaginylendo-peptidase,Arginylendo-peptidase, Achromobacter protease I, Trypsin, Staphlococcusaureus V8 protease, Endoproteinase Asp-N, or Endoproteinase Lys-C underconventional conditions to result in cleavage at specific amino acidresidues. Asparaginylendo-peptidase can cleave specifically on thecarboxyl side of the asparagine residues present within the polypeptidesof the invention. Arginylendo-peptidase can cleave specifically on thecarboxyl side of the arginine residues present within thesepolypeptides. Achromobacter protease I can cleave specifically on thecarboxyl side of the lysine residues present within the polypeptides(Sakiyama and Nakat, U.S. Pat. No. 5,248,599; T. Masaki et al., Biochim.Biophys. Acta 660:44-50 (1981); T. Masaki et al., Biochim. Biophys. Acta660:51-55 (1981)). Trypsin can cleave specifically on the carboxyl sideof the arginine and lysine residues present within polypeptides of theinvention. Enzymatic fragmentation may also occur with a protease thatcleaves at multiple amino acid residues. For example, Staphlococcusaureus V8 protease can cleave specifically on the carboxyl side of theaspartic and glutamic acid residues present within polypeptides (D. W.Cleveland, J. Biol. Chem. 3:1102-1106 (1977)). Endoproteinase Asp-N cancleave specifically on the amino side of the asparagine residues presentwithin polypeptides. Endoproteinase Lys-C can cleave specifically on thecarboxyl side of the lysine residues present within polypeptides of theinvention. Other enzymatic and chemical treatments can likewise be usedto specifically fragment these polypeptides into a unique set ofspecific peptides.

[0211] Of course, the peptides and fragments of the polypeptides of theinvention can also be produced by conventional recombinant processes andsynthetic processes well known in the art. With regard to recombinantprocesses, the polypeptides and peptide fragments encompassed byinvention can have variable molecular weights, depending upon the hostcell in which they are expressed. Glycosylation of polypeptides andpeptide fragments of the invention in various cell types can result invariations of the molecular weight of these pieces, depending upon theextent of modification. The size of these pieces can be mostheterogeneous with fragments of polypeptide derived from theextracellular portion of the polypeptide. Consistent polypeptides andpeptide fragments can be obtained by using polypeptides derived entirelyfrom the transmembrane and cytoplasmic regions, pretreating withN-glycanase to remove glycosylation, or expressing the polypeptides inbacterial hosts.

[0212] The molecular weight of these polypeptides can also be varied byfusing additional peptide sequences to both the amino and carboxylterminal ends of polypeptides of the invention. Fusions of additionalpeptide sequences at the amino and carboxyl terminal ends ofpolypeptides of the invention can be used to enhance expression of thesepolypeptides or aid in the purification of the protein. In addition,fusions of additional peptide sequences at the amino and carboxylterminal ends of polypeptides of the invention will alter some, butusually not all, of the fragmented peptides of the polypeptidesgenerated by enzymatic or chemical treatment. Of course, mutations canbe introduced into polypeptides of the invention using routine and knowntechniques of molecular biology. For example, a mutation can be designedso as to eliminate a site of proteolytic cleavage by a specific enzymeor a site of cleavage by a specific chemically induced fragmentationprocedure. The elimination of the site will alter the peptidefingerprint of polypeptides of the invention upon fragmentation with thespecific enzyme or chemical procedure.

[0213] The polypeptides and the resultant fragmented peptides can beanalyzed by methods including sedimentation, electrophoresis,chromatography, and mass spectrometry to determine their molecularweights. Because the unique amino acid sequence of each piece specifiesa molecular weight, these pieces can thereafter serve as molecularweight markers using such analysis techniques to assist in thedetermination of the molecular weight of an unknown protein,polypeptides or fragments thereof. The molecular weight markers of theinvention serve particularly well as molecular weight markers for theestimation of the apparent molecular weight of proteins that havesimilar apparent molecular weights and, consequently, allow increasedaccuracy in the determination of apparent molecular weight of proteins.

[0214] When the invention relates to the use of fragmented peptidemolecular weight markers, those markers are preferably at least 10 aminoacids in size. More preferably, these fragmented peptide molecularweight markers are between 10 and 100 amino acids in size. Even morepreferable are fragmented peptide molecular weight markers between 10and 50 amino acids in size and especially between 10 and 35 amino acidsin size. Most preferable are fragmented peptide molecular weight markersbetween 10 and 20 amino acids in size.

[0215] Among the methods for determining molecular weight aresedimentation, gel electrophoresis, chromatography, and massspectrometry. A particularly preferred embodiment is denaturingpolyacrylamide gel electrophoresis (U. K. Laemmli, Nature 227:680-685(1970)). Conventionally, the method uses two separate lanes of a gelcontaining sodium dodecyl sulfate and a concentration of acrylamidebetween 6-20%. The ability to simultaneously resolve the marker and thesample under identical conditions allows for increased accuracy. It isunderstood, of course, that many different techniques can be used forthe determination of the molecular weight of an unknown protein usingpolypeptides of the invention, and that this embodiment in no way limitsthe scope of the invention.

[0216] Each unglycosylated polypeptide or fragment thereof has a pi thatis intrinsically determined by its unique amino acid sequence (which pIcan be estimated by the skilled artisan using any of the computerprograms designed to predict pI values currently available, calculatedusing any well-known amino acid pKa table, or measured empirically).Therefore these polypeptides and fragments thereof can serve as specificmarkers to assist in the determination of the isoelectric point of anunknown protein, polypeptide, or fragmented peptide using techniquessuch as isoelectric focusing. These polypeptide or fragmented peptidemarkers serve particularly well for the estimation of apparentisoelectric points of unknown proteins that have apparent isoelectricpoints close to that of the polypeptide or fragmented peptide markers ofthe invention.

[0217] The technique of isoelectric focusing can be further combinedwith other techniques such as gel electrophoresis to simultaneouslyseparate a protein on the basis of molecular weight and charge. Theability to simultaneously resolve these polypeptide or fragmentedpeptide markers and the unknown protein under identical conditionsallows for increased accuracy in the determination of the apparentisoelectric point of the unknown protein. This is of particular interestin techniques, such as two dimensional electrophoresis (T. D. Brock andM. T. Madigan, Biology of Microorganisms 76-77, Prentice Hall, 6d ed.(1991)), where the nature of the procedure dictates that any markersshould be resolved simultaneously with the unknown protein. In addition,with such methods, these polypeptides and fragmented peptides thereofcan assist in the determination of both the isoelectric point andmolecular weight of an unknown protein or fragmented peptide.

[0218] Polypeptides and fragmented peptides can be visualized using twodifferent methods that allow a discrimination between the unknownprotein and the molecular weight markers. In one embodiment, thepolypeptide and fragmented peptide molecular weight markers of theinvention can be visualized using antibodies generated against thesemarkers and conventional immunoblotting techniques. This detection isperformed under conventional conditions that do not result in thedetection of the unknown protein. It is understood that it may not bepossible to generate antibodies against all polypeptide fragments of theinvention, since small peptides may not contain immunogenic epitopes. Itis further understood that not all antibodies will work in this assay;however, those antibodies which are able to bind polypeptides andfragments of the invention can be readily determined using conventionaltechniques.

[0219] The unknown protein is also visualized by using a conventionalstaining procedure. The molar excess of unknown protein to polypeptideor fragmented peptide molecular weight markers of the invention is suchthat the conventional staining procedure predominantly detects theunknown protein. The level of these polypeptide or fragmented peptidemolecular weight markers is such as to allow little or no detection ofthese markers by the conventional staining method. The preferred molarexcess of unknown protein to polypeptide molecular weight markers of theinvention is between 2 and 100,000 fold. More preferably, the preferredmolar excess of unknown protein to these polypeptide molecular weightmarkers is between 10 and 10,000 fold and especially between 100 and1,000 fold.

[0220] It is understood of course that many techniques can be used forthe determination and detection of molecular weight and isoelectricpoint of an unknown protein, polypeptides, and fragmented peptidesthereof using these polypeptide molecular weight markers and peptidefragments thereof and that these embodiments in no way limit the scopeof the invention.

[0221] In another embodiment, the analysis of the progressivefragmentation of the polypeptides of the invention into specificpeptides (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106 (1977)),such as by altering the time or temperature of the fragmentationreaction, can be used as a control for the extent of cleavage of anunknown protein. For example, cleavage of the same amount of polypeptideand unknown protein under identical conditions can allow for a directcomparison of the extent of fragmentation. Conditions that result in thecomplete fragmentation of the polypeptide can also result in completefragmentation of the unknown protein.

[0222] As to the specific use of the polypeptides and fragmentedpeptides of the invention as molecular weight markers, the fragmentationof the polypeptide of SEQ ID NO:2 with cyanogen bromide generates aunique set of fragmented peptide molecular weight markers. Thedistribution of methionine residues determines the number of amino acidsin each peptide and the unique amino acid composition of each peptidedetermines its molecular weight.

[0223] In addition, the preferred purified polypeptide of the invention(SEQ ID NO:2) has an observed molecular weight of approximately 21,000Daltons.

[0224] Where an intact protein is used, the use of these polypeptidemolecular weight markers allows increased accuracy in the determinationof apparent molecular weight of proteins that have apparent molecularweights close to 21,000 Daltons. Where fragments are used, there isincreased accuracy in determining molecular weight over the range of themolecular weights of the fragment.

[0225] Finally, as to the kits that are encompassed by the invention,the constituents of such kits can be varied, but typically contain thepolypeptide and fragmented peptide molecular weight markers. Also, suchkits can contain the polypeptides wherein a site necessary forfragmentation has been removed. Furthermore, the kits can containreagents for the specific cleavage of the polypeptide and the unknownprotein by chemical or enzymatic cleavage. Kits can further containantibodies directed against polypeptides or fragments thereof of theinvention.

[0226] Identification of Unknown Proteins

[0227] As set forth above, a polypeptide or peptide fingerprint can beentered into or compared to a database of known proteins to assist inthe identification of the unknown protein using mass spectrometry (W. J.Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015 (1993); D. Fenyoet al., Electrophoresis 19:998-1005 (1998)). A variety of computersoftware programs to facilitate these comparisons are accessible via theInternet, such as Protein Prospector (Internet site:prospector.uscf.edu), MultiIdent (Internet site:www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site:www.mann.embl-heiedelberg.de . . . deSearch/FR_PeptideSearch Form.html),and ProFound (Internet site:www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programsallow the user to specify the cleavage agent and the molecular weightsof the fragmented peptides within a designated tolerance. The programscompare these molecular weights to protein databases to assist indetermining the identity of the unknown protein.

[0228] In addition, a polypeptide or peptide digest can be sequencedusing tandem mass spectrometry (MS/MS) and the resulting sequencesearched against databases (J. K. Eng, et al., J. Am. Soc. Mass Spec.5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem. 66:4390-4399 (1994);J. A. Taylor and R. S. Johnson, Rapid Comm. Mass Spec. 11:1067-1075(1997)). Searching programs that can be used in this process exist onthe Internet, such as Lutefisk 97 (Internet site:www.lsbc.com:70/Lutefisk97.html), and the Protein Prospector, PeptideSearch and ProFound programs described above. Therefore, adding thesequence of a gene and its predicted protein sequence and peptidefragments to a sequence database can aid in the identification ofunknown proteins using tandem mass spectrometry.

[0229] Antibodies

[0230] Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

[0231] These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9, Garland Publishing Inc., 2nd ed. (1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hinderances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14, Garland Publishing Inc., 2nded. (1996)). Epitopes may be identified by any of the methods known inthe art.

[0232] Thus, one aspect of the present invention relates to theantigenic epitopes of the polypeptides of the invention. Such epitopesare useful for raising antibodies, in particular monoclonal antibodies,as described in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supematants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

[0233] As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,(Kennet et al., Monoclonal Antibodies, Hybridomas: A New Dimension inBiological Analyses, eds., Plenum Press, New York (1980); and Harlow andLand, Antibodies: A Laboratory Manual, eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1988)).

[0234] Hybridoma cell lines that produce monoclonal antibodies specificfor the polypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide; harvesting spleen cells from the immunizedanimal; fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells; and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies may be recovered by conventional techniques.

[0235] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., humanized versions of murine monoclonalantibodies. Such humanized antibodies may be prepared by knowntechniques and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a humanizedmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise the antigen binding site of a murine monoclonal antibody and avariable region fragment (lacking the antigen-binding site) derived froma human antibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in (Riechlnannet al., Nature 332:323 (1988), Liu et al., PNAS84:3439 (1987), Larricket al., Bio/Technology 7:934 (1989), and Winter and Harris, TIPS 14:139(May 1993)). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein.

[0236] Antigen-binding fragments of the antibodies, which may beproduced by conventional techniques, are also encompassed by the presentinvention. Examples of such fragments include, but are not limited to,Fab and F(ab′)₂ fragments. Antibody fragments and derivatives producedby genetic engineering techniques are also provided.

[0237] In one embodiment, the antibodies are specific for thepolypeptides of the present invention and do not cross-react with otherproteins. Screening procedures by which such antibodies may beidentified are well known, and may involve immunoaffinitychromatography, for example.

[0238] Uses Thereof

[0239] The antibodies of the invention can be used in assays to detectthe presence of the polypeptides or fragments of the invention, eitherin vitro or in vivo. The antibodies also may be employed in purifyingpolypeptides or fragments of the invention by immunoaffinitychromatography.

[0240] Those antibodies that additionally can block binding of thepolypeptides of the invention to TSLP receptors may be used to inhibit abiological activity that results from such binding. Such blockingantibodies may be identified using any suitable assay procedure, such asby testing antibodies for the ability to inhibit binding of TSLP tocertain cells expressing the TSLP receptors. Examples of such cells arethe B and T lymphoid cell lines 70Z/3 and 7B9, respectively.Alternatively, blocking antibodies may be identified in assays for theability to inhibit a biological effect that results from binding of TSLPto TSLP receptors on target cells. Antibodies may be assayed for theability to inhibit TSLP-mediated lysis of cells expressing TSLPreceptors, for example.

[0241] Such an antibody may be employed in an in vitro procedure, oradministered in vivo to inhibit a biological activity mediated by theentity that generated the antibody. Disorders caused or exacerbated(directly or indirectly) by the interaction of TSLP with cell surfaceTSLP receptors thus may be treated. A therapeutic method involves invivo administration of a blocking antibody to a mammal in an amounteffective in inhibiting TSLP-mediated biological activity. Monoclonalantibodies are generally preferred for use in such therapeutic methods.In one embodiment, an antigen-binding antibody fragment is employed.

[0242] Antibodies may be screened for agonistic (ie., ligand-mimicking)properties. Such antibodies, upon binding to cell surface TSLPreceptors, induce biological effects (e.g., transduction of biologicalsignals) similar to the biological effects induced when TSLP binds tocell surface TSLP receptors. Agonistic antibodies may be used to induceB lineage or T lineage cell proliferation.

[0243] Compositions comprising an antibody that is directed againsthuman TSLP, and a physiologically acceptable diluent, excipient, orcarrier, are provided herein. Suitable components of such compositionsare as described above for compositions containing human TSLP proteins.

[0244] Also provided herein are conjugates comprising a detectable(e.g., diagnostic) or therapeutic agent, attached to the antibody.Examples of such agents are presented above. The conjugates find use inin vitro or in vivo procedures.

[0245] The following examples are provided to further illustrateparticular embodiments of the invention, and are not to be construed aslimiting the scope of the present invention.

EXAMPLE 1 Isolation of the Nucleic Acid

[0246] Human TSLP nucleic acid sequence was obtained by sequencing ESTIMAGE clone 1407260, accession #AA889581. This sequence suggested, incomparison to the murine TSLP sequence, that the EST clone was a partialclone. A number of cDNA libraries were screened with internal primers todetermine a source of cDNA that could be used to obtain the missing 3′end of the TSLP cDNA clone. After 60 cycles of PCR using two internalprimers of human TSLP sequence, the following cDNA libraries werepositive for TSLP sequences: human testis, human foreskin fibroblasts,and fetal brain (weakly positive); while MoT, HS431, bone marrow, HPT4,HBT3, W126, Hut102, PBT, Sk Hep, human dermal fibroblast, Raji, humanplacenta, and KB libraries were all negative.

[0247] Using PCR on the human testis λgt10 library with an internal TSLPprimer and a λgt10 vector primer, two clones (19E and 19F) withsequences identical to internal human TSLP sequences were isolated. Bothclones had identical 5′ ends but different length 3′ ends. The coding aswell as the non-coding sequences of clone 19E were identical to clone19F; these clones differed in the length of the 3′ non-coding region,where clone 19F was about 34 bp longer than 19E. Therefore, sequencesfrom 19F were used to complete the 3′ coding sequence of the human TSLPprotein. This allowed for the identification of the C-terminal 15 aminoacids not present in the EST. PCR was conducted according toconventional procedures.

EXAMPLE 2 Purification of TSLP Polypeptide

[0248] TSLP-Specific ELISA:

[0249] Serial dilutions of TSLP-containing samples (in 50 mM NaHCO₃,brought to pH 9 with NaOH) are coated onto Linbro/Titertek 96 well flatbottom E.I.A. microtitration plates (ICN Biomedicals Inc., Aurora, Ohio)at 100:1/well. After incubation at 4° C. for 16 hours, the wells arewashed six times with 200:1 PBS containing 0.05% Tween-20 (P3S-Tween).The wells are then incubated with FLAG®-TSLP receptor at 1 μg/ml inPBS-Tween with 5% fetal calf serum (FCS) for 90 minutes (100:1 perwell), followed by washing as above. Next, each well is incubated withthe anti-FLAG® (monoclonal antibody M2 at 1 μg/ml in PBS-Tweencontaining 5% FCS for 90 minutes (100:1 per well), followed by washingas above. Subsequently, wells are incubated with a polyclonal goatanti-mIgG1-specific horseradish peroxidase-conjugated antibody (a 1:5000dilution of the commercial stock in PBS-Tween containing 5% FCS) for 90minutes (100:1 per well). The HRP-conjugated antibody is obtained fromSouthern Biotechnology Associates, Inc., Birmingham, Ala. Wells then arewashed six times, as above.

[0250] For development of the ELISA, a substrate mix [100:1 per well ofa 1:1 premix of the TMB Peroxidase Substrate and Peroxidase Solution B(Kirkegaard Perry Laboratories, Gaithersburg, Md.)] is added to thewells. After sufficient color reaction, the enzymatic reaction isterminated by addition of 2 N H₂SO₄ (50:1 per well). Color intensity(indicating TSLP-TSLP receptor binding) is determined by measuringextinction at 450 nm on a V Max plate reader (Molecular Devices,Sunnyvale, Calif.).

EXAMPLE 3 Amino Acid Sequence

[0251] The amino acid sequence of human TSLP was determined bytranslation of the complete human TSLP nucleotide sequence. The readingframe chosen was based on the homology of human TSLP with murine TSLP.

EXAMPLE 4 DNA and Amino Acid Sequences

[0252] The human TSLP nucleic acid sequence was determined by standarddouble stranded sequencing of the composite sequence of EST IMAGE clone1407260, accession #AA889581, and the additional 3′ sequence from clone19F.

[0253] The nucleotide sequence of the isolated human TSLP DNA and theamino acid sequence encoded thereby, are presented in SEQ ID NOs: 1 and2. The sequence of the entire human TSLP DNA fragment isolated by PCRcorresponds to nucleotides 1 to 767 of SEQ ID NO: 1, which encode aminoacids 1 to 159 of SEQ ID NO:2.

[0254] The amino acid sequence in SEQ ID NO:2 bears significantsimilarity (49%) and identity (43%) to murine TSLP and weak homology toIL-7.

EXAMPLE 5 Monoclonal Antibodies that Bind TSLP

[0255] This example illustrates a method for preparing monoclonalantibodies that bind TSLP. Suitable immunogens that may be employed ingenerating such antibodies include, but are not limited to, purifiedhuman TSLP polypeptide or an immunogenic fragment thereof such as theextracellular domain, or fusion proteins containing human TSLP (e.g., asoluble TSLP/Fc fusion protein).

[0256] Purified human TSLP can be used to generate monoclonal antibodiesimmunoreactive therewith, using conventional techniques such as thosedescribed in U.S. Pat. No. 4,411,993. Briefly, mice are immunized withhuman TSLP immunogen emulsified in complete Freund's adjuvant, andinjected in amounts ranging from 10-100 μg subcutaneously orintraperitoneally. Ten to twelve days later, the immunized animals areboosted with additional human TSLP emulsified in incomplete Freund'sadjuvant. Mice are periodically boosted thereafter on a weekly tobi-weekly immunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision to test for TSLP antibodiesby dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay) orinhibition of TSLP receptor binding.

[0257] Following detection of an appropriate antibody titer, positiveanimals are provided one last intravenous injection of human TSLP insaline. Three to four days later, the animals are sacrificed, spleencells harvested, and spleen cells are fused to a murine myeloma cellline, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusionsgenerate hybridoma cells, which are plated in multiple microtiter platesin a HAT (hypoxanthine, aminopterin and thymidine) selective medium toinhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

[0258] The hybridoma cells are screened by ELISA for reactivity againstpurified TSLP by adaptations of to the techniques disclosed in (Engvallet al., Immunochem. 8:871 (1971)) and in U.S. Pat. No. 4,703,004. Apreferred screening technique is the antibody capture techniquedescribed in (Beckmann et al., J. Immunol. 144:4212 (1990)). Positivehybridoma cells can be injected intraperitoneally into syngeneic BALB/cmice to produce ascites containing high concentrations of anti-TSLPmonoclonal antibodies. Alternatively, hybridoma cells can be grown invitro in flasks or roller bottles by various techniques. Monoclonalantibodies produced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to Protein A orProtein G can also be used, as can affinity chromatography based uponbinding to TSLP.

EXAMPLE 6 Northern Blot Analysis

[0259] The tissue distribution of human TSLP mRNA was investigated byNorthern blot analysis, as follows. An aliquot of a radiolabeled probewas added to two different human multiple tissue Northern blots(Clontech, Palo Alto, Calif.; Biochain, Palo Alto, Calif.). The blotswere hybridized in 10× Denhardts, 50 mM Tris pH 7.5, 900 mM NaCl, 0.1%Na pyrophosphate, 1% SDS, 200 μg/mL salmon spern DNA. Hybridization wasconducted overnight at 63° C. in 50% formamide as previously described(March et al., Nature 315:641-647 (1985)). The blots then were washedwith 2×SSC, 0.1% SDS at 68° C. for 30 minutes

[0260] A single transcript of 1.4 kilobases (kb) was present in heart,lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus,prostate, testes, ovary, small intestine, colon. Negative tissues werebrain, placenta, and peripheral blood leukocytes. The cells and tissueswith the highest levels of TSLP mRNA are heart, liver, prostate, andtestes, as shown by comparison to control probing with aβ-actin-specific probe.

EXAMPLE 7 Binding Assay

[0261] Full length human TSLP can be expressed and tested for theability to bind TSLP receptors. The binding assay can be conducted asfollows.

[0262] A fusion protein comprising a leucine zipper peptide fused to theN-terminus of a soluble human TSLP polypeptide (LZ-TSLP) is employed inthe assay. An expression construct is prepared, essentially as describedfor preparation of the FLAG®(TSLP) expression construct in (Wiley etal., Immunity, 3:673-682 (1995)); hereby incorporated by reference),except that DNA encoding the FLAG® peptide was replaced with a sequenceencoding a modified leucine zipper that allows for trimerization. Theconstruct, in expression vector pDC409, encodes a leader sequencederived from human cytomegalovirus, followed by the leucine zippermoiety fused to the N-terminus of a soluble human TSLP polypeptide. TheLZ-TSLP is expressed in CHO cells, and purified from the culturesupernatant.

[0263] The expression vector designated pDC409 is a mammalian expressionvector derived from the pDC406 vector described in (McMahan et al., EMBOJ. 10:2821-2832 (1991)) hereby incorporated by reference). Featuresadded to pDC409 (compared to pDC406) include additional uniquerestriction sites in the multiple cloning site (mcs); three stop codons(one in each reading frame) positioned downstream of the mcs; and a T7polymerase promoter, downstream of the mcs, that facilitates sequencingof DNA inserted into the mcs.

[0264] For expression of full length human TSLP protein, the entirecoding region (i.e., the DNA sequence presented in SEQ ID NO:1) isamplified by polymerase chain reaction (PCR). The template employed inthe PCR is the cDNA clone isolated from a human testis cDNA library, asdescribed in Example 1. The isolated and amplified DNA is inserted intothe expression vector pDC409, to yield a construct designatedpDC409-TSLP.

[0265] LZ-TSLP polypeptide is employed to test the ability to bind tohost cells expressing recombinant or endogenous TSLP receptors, asdiscussed above. Cells expressing TSLP receptor are cultured in DMEMsupplemented with 10% fetal bovine serum, penicillin, streptomycin, andglutamine. Cells are incubated with LZ-TSLP (5 mg/ml) for about 1 hour.Following incubation, the cells are washed to remove unbound LZ-TSLP andincubated with a biotinylated anti-LZ monoclonal antibody (5 mg/ml), andphycoerythrin-conjugated streptavidin (1:400), before analysis byfluorescence-activated cell scanning (FACS). The cytometric analysis wasconducted on a FACscan (Beckton Dickinson, San Jose, Calif.).

[0266] The cells expressing TSLP receptors showed significantly enhancedbinding of LZ-TSLP, compared to the control cells not expressing TSLPreceptors.

EXAMPLE 8 Induction of T Cell Growth from Bone Marrow By TSLP and IL-7

[0267] Human TSLP, in combination with IL-7, induces the outgrowth of Tcells from human bone marrow.

[0268] Human bone marrow-derived mononuclear cells (BM MNC) wereisolated by centrifugation of whole bone marrow over Ficoll. BM MNC werecultured in McCoy's media supplemented with 10% fetal bovine serum, andamino acid and vitamin supplements, at a concentration ranging between4.5-10×10⁵ cells/ml in a total volume of 6 or 7 ml per flask (T25) HumanTSLP (20 ng/ml) and other cytokines, i.e., 1L-7, SLF (i.e., steel factoror stem cell factor, or mast cell growth factor), or flt3L, either aloneor in combination, were added to the cultures at day 0. After 14 daysand weekly thereafter, half the culture was removed for counting. Freshmedia and cytokines were added to the cultures to return the totalvolume to 6 or 7 ml.

[0269] Harvested cells were also analyzed via flow cytometry fourteendays after culture and weekly thereafter, using antibodies specific forcell surface antigens The antibodies used were specific for T cellantigens (i.e., the αβ T cell receptor, γδ T cell receptor, and CD3), Bcell antigens (i.e., CD19 and surface IgM), Natural Killer cell antigens(i.e., CD56), monocyte antigens (i.e., CD 14), and granulocyte antigens(i.e., CD15).

[0270] Addition of human TSLP and IL-7 to BM MNC cultures inducedcellular growth as indicated in Table 1. At day 0, approximately 5% ofBM MNC were T cells. After 2 weeks of culture with TSLP and IL-7, thecultures consisted of 70% CD3⁺ T cells. At day 21, 86% of the cells wereCD3⁺ T cells. The cultures contained predominantly T cells until thetermination of the experiment at day 42. TABLE I Total Cell Yield (×10⁵) Cumu- la- Treatment Day 0 Day 14 Day 21 Day 28 Day 42 tive 13.5Media 6 1.1 0.4 0.9 8.4 TSLP 3.9 2.1 1 2.9 9.9 IL-7 4.2 7.4 4.4 4.6 20.6IL-7 + TSLP 10.3 12.1 17.2 7.5 47.1 SLF 3.7 4.3 1.1 0.9 10 SLF + TSLP5.4 6.9 1 1.6 14.9 flt3L 6.3 2.3 2.8 1.8 13.2 flt3L + TSLP 7.7 4.7 2.73.1 18.2

[0271] In another set of experiments, three separate batches of humanTSLP tagged with His/FLAG® (TSLP 7489, TSLP 7811, or TSLP 7812) weretested alone or in combination with IL-7 for the ability to affect cellsurvival and expansion. BM MNC cultures were obtained from two separate,fresh bone marrow samples and seeded at a concentration of either 5×10⁵cells/ml (Group 1) or 10×10⁵ cells/ml (Group 2). His/FLAG®-tagged TSLP(20 mg/ml) and IL-7 were added to cultures as described above. TSLPcombined with IL-7 resulted in expansion of BM MNC cultures as indicatedin Table 2 (bone marrow sample 1) and Table 3 (bone marrow sample 2). Byday 21, 80% of the expanded cell population consisted of CD4⁺ αβ⁺ orCD8⁺ αβ⁺ T cells. In four of the cultures treated with IL-7 and TSLP,cells expanded at such a cultures contained predominantly T cells untilthe termination of the experiments at 4-5 weeks. TABLE 2 Total CellYield (× 10⁵) Cumu- la- Treatment Day 0 Day 14 Day 21 Day 28 Day 35 tiveGroup 1 17.5 (5 ×10⁵) Media 4 1.3 1.4 ND* 6.7 IL-7 8.4 6.5 7.1 ND* 22TSLP 7489 4.4 1.5 1.2 ND* 7.1 TSLP 7811 5.2 1.7 1.2 ND* 8.1 TSLP 78122.8 1.4 2.3 ND* 6.5 IL-7 + T7489 12.4 9.1 8.3 ND* 29.8 IL-7 + T7811 10.55.3 8.4 ND* 24.2 IL-7 + T7812 9.7 6.5 4.7 ND* 20.9 Group 2 35 (10 × 10⁵)Media 6.6 3.1 2.2 ND* 11.9 IL-7 14.8 10.1 3.7 ND* 32.3 TSLP 7489 11.53.3 2.9 ND* 17.7 TSLP 7811 13.3 2.8 3.1 ND* 19.2 TSLP 7812 13 3.2 2.6ND* 18.8 IL-7 + T7489 25.6 17.7 8 10.9 62.2 IL-7 + T7811 18.8 16.8 1015.7 61.3 IL-7 + T7812 22.4 13.5 10.4 11.6 57.9

[0272] TABLE 3 Total Cell Yield (× 10⁵) Treat- Day Day Day Day Day Dayment 0 14 21 23 28 35 Cumulative Group 1 17.5 (5 × 10⁵) Media 3.1 0.9ND* 0.8 ND* 4.8 IL-7 3.8 8.9 ND* 8 ND* 20.7 TSLP 7489 3 1.1 ND* 0.8 ND*4.9 TSLP 7811 2.6 1.3 ND* ND* ND* 3.9 TSLP 7812 3.8 1.2 ND* 0.9 ND* 5.9IL-7 + T7489 8.9 80 39.4 18.2 21 167.5 IL-7 + T7811 6.2 12.5 ND* 16.714.3 49.7 IL-7 + T7812 7.1 14.5 ND* 11.1 11.6 44.3 Group 2 35 (10 × 10⁵)Media 6.6 1.9 ND* 1.8 ND* 10.3 IL-7 10.7 19 ND* 16.5 29.2 75.4 TSLP 74896.8 3.2 ND* 3.3 ND* 13.3 TSLP 7811 8.7 3.3 ND* 3.4 ND* 15.4 TSLP 78127.1 3.1 ND* 2.7 ND* 12.9 IL-7 + T7489 18.1 31.4 20 16.7 20.4 106.6IL-7 + T7811 13.9 26.2 46.8 17.9 19.2 124 IL-7 + T7812 15.1 24.4 88.420.6 26.6 175.1

[0273] The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments of the inventionand should not be construed to limit the scope of the invention. Theskilled artisan readily recognizes that many other embodiments areencompassed by the invention.

1 5 1 743 DNA Homo sapiens 1 gcagccagaa agctctggag catcagggag actccaacttaaggcaacag catgggtgaa 60 taagggcttc ctgtggactg gcaatgagag gcaaaacctggtgcttgagc actggcccct 120 aaggcaggcc ttacagatct cttacactcg tggtgggaagagtttagtgt gaaactgggg 180 tggaattggg tgtccacgta tgttcccttt tgccttactatatgttctgt cagtttcttt 240 caggaaaatc ttcatcttac aacttgtagg gctggtgttaacttacgact tcactaactg 300 tgactttgag aagattaaag cagcctatct cagtactatttctaaagacc tgattacata 360 tatgagtggg accaaaagta ccgagttcaa caacaccgtctcttgtagca atcggccaca 420 ttgccttact gaaatccaga gcctaacctt caatcccaccgccggctgcg cgtcgctcgc 480 caaagaaatg ttcgccatga aaactaaggc tgccttagctatctggtgcc caggctattc 540 ggaaactcag ataaatgcta ctcaggcaat gaagaagaggagaaaaagga aagtcacaac 600 caataaatgt ctggaacaag tgtcacaatt acaaggattgtggcgtcgct tcaatcgacc 660 tttactgaaa caacagtaaa ccatctttat tatggtcatatttcacagcc caaaataaat 720 catctttatt aagtaaaaaa aaa 743 2 159 PRT Homosapiens 2 Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe ArgLys 1 5 10 15 Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr AspPhe Thr 20 25 30 Asn Cys Asp Phe Glu Lys Ile Lys Ala Ala Tyr Leu Ser ThrIle Ser 35 40 45 Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser Thr GluPhe Asn 50 55 60 Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu Thr GluIle Gln 65 70 75 80 Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser LeuAla Lys Glu 85 90 95 Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile TrpCys Pro Gly 100 105 110 Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala MetLys Lys Arg Arg 115 120 125 Lys Arg Lys Val Thr Thr Asn Lys Cys Leu GluGln Val Ser Gln Leu 130 135 140 Gln Gly Leu Trp Arg Arg Phe Asn Arg ProLeu Leu Lys Gln Gln 145 150 155 3 8 PRT Artificial Sequence Descriptionof Artificial Sequence antigenic peptide used in fusion proteins 3 AspTyr Lys Asp Asp Asp Asp Lys 1 5 4 27 PRT Artificial Sequence Descriptionof Artificial Sequence leucine zipper polypeptide 4 Pro Asp Val Ala SerLeu Arg Gln Gln Val Glu Ala Leu Gln Gly Gln 1 5 10 15 Val Gln His LeuGln Ala Ala Phe Ser Gln Tyr 20 25 5 33 PRT Artificial SequenceDescription of Artificial Sequence leucine zipper polypeptide 5 Arg MetLys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile 1 5 10 15 TyrHis Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu 20 25 30 Arg

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of: (a) the DNA sequence of SEQ ID NO:1; (b) anisolated nucleic acid molecule encoding an amino acid sequencecomprising the sequence of SEQ ID NO:2; (c) an isolated nucleic acidmolecule that hybridizes to either strand of a denatured,double-stranded DNA comprising the nucleic acid sequence of (a) or (b)under conditions of moderate stringency in 50% formamide and 6×SSC, at42° C. with washing conditions of 60° C., 0.5×SSC, 0.1% SDS; (d) anisolated nucleic acid molecule derived by in vitro mutagenesis from SEQID NO:1; (e) an isolated nucleic acid molecule degenerate from SEQ IDNO:1 as a result of the genetic code; and (f) an isolated nucleic acidmolecule selected from the group consisting of human TSLP DNA, anallelic variant of human TSLP DNA, and a species homolog of TSLP DNA. 2.The nucleic acid molecule of claim 1 comprising the DNA sequence of SEQID NO:1.
 3. A recombinant vector that directs the expression of thenucleic acid molecule of claim
 1. 4. An isolated polypeptide encoded bythe nucleic acid molecule of claim
 1. 5. An isolated polypeptideaccording to claim 4 having a molecular weight of approximately 21,000Daltons as determined by SDS-PAGE.
 6. An isolated polypeptide accordingto claim 4 in non-glycosylated form.
 7. An isolated antibody that bindsto a polypeptide of claim
 4. 8. An antibody according to claim 7,wherein the antibody is a monoclonal antibody.
 9. A purified TSLPpolypeptide selected from the group consisting of: a) the TSLPpolypeptide of SEQ ID NO:2; and b) a fragment of the polypeptide of (a),wherein said fragment is capable of binding TSLP receptors.
 10. Anisolated TSLP polypeptide comprising an amino acid sequence that is atleast 80% identical to the amino acid sequence presented in SEQ ID NO:2.11. A TSLP polypeptide of claim 9, wherein said polypeptide comprisesthe amino acid sequence of SEQ ID NO:2.
 12. A composition comprising apolypeptide of claim 4, and a physiologically acceptable diluent,excipient, or carrier.
 13. A host cell transfected or transduced withthe vector of claim
 3. 14. A method for the production of TSLPpolypeptide comprising culturing the host cell of claim 13 underconditions promoting expression, and recovering the polypeptide from theculture medium.
 15. The method of claim 14, wherein the host cell isselected from the group consisting of bacterial cells, yeast cells,plant cells, and animal cells.
 16. A method of stimulating lymphocyteproliferation, comprising incubating lymphocytes with the polypeptideaccording to claim
 4. 17. The method of claim 16, further comprisingincubating the lymphocytes with IL-7.
 18. The method of claim 16,further comprising incubating the lymphocytes with a cytokine selectedfrom the group consisting of Steel Factor, Stem Cell Factor, Mast CellGrowth Factor or flt3-Ligand.
 19. A method of stimulating lymphocytedevelopment or lymphopoiesis comprising incubating progenitor cells withthe polypeptide according to claim
 4. 20. The method of claim 19,wherein the progenitor cells are bone marrow-derived mononuclear cells.21. The method of claim 20, further comprising incubating the bonemarrow-derived mononuclear cells with IL-7.
 22. A method of stimulatinglymphocyte proliferation, comprising incubating lymphocytes with thepolypeptide according to claim 4 and IL-7.
 23. A method of stimulatinglymphocyte development or lymphopoiesis comprising incubating progenitorcells with the polypeptide according to claim 4 and IL-7.